Anti-cd154 antibodies

ABSTRACT

The present invention provides peptides, and fragments thereof, and antibodies, or fragments thereof comprising the same, wherein the peptide comprises at least one amino acid substitution compared to wild type 5c8 antibody. The present invention also provides compositions and methods of treating CD154-related diseases or disorders in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 11/572,647 which is a U.S. national phase application under 35U.S.C. §371 of International Application Serial No. PCT/US2005/026320filed Jul. 26, 2005, which claims priority to U.S. provisionalapplication Ser. No. 60/591,337 filed Jul. 26, 2004, each of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 10, 2013, isnamed B2047-7072US_SL.txt and is 84,467 bytes in size.

FIELD OF THE INVENTION

The present invention is directed, in part, to anti-CD154 antibodies andfragments thereof that comprise at least one substituted amino acid inthe variable region of either or both the light and heavy chain, and tocompositions comprising such antibodies or fragments thereof, and tomethods of using the antibodies or fragments thereof.

BACKGROUND OF THE INVENTION

The generation of humoral and cell-mediated immunity is orchestrated bythe interaction of activated helper T cells with antigen-presentingcells (“APCs”) and effector T cells. Activation of the helper T cells isnot only dependent on the interaction of the antigen-specific T-cellreceptor (“TCR”) with its cognate peptide-MHC ligand, but also requiresthe coordinate binding and activation by a number of cell adhesion andcostimulatory molecules (Salazar-Fontana et al., Curr. Opin. Hemat.,2001, 8, 5).

A critical costimulatory molecule is CD154 (also known as CD40 ligand,CD40L, gp39, T-BAM, T-Cell Activating Molecule, TRAP), a Type IItransmembrane protein that is expressed in an activation-dependent,temporally-restricted, manner on the surface of CD4+ T cells. CD154 isalso expressed, following activation, on a subset of CD8+ T cells,basophils, mast cells, eosinophils, natural killer cells, B cells,macrophages, dendritic cells and platelets. The CD154 counter-receptor,CD40, is a Type I membrane protein that is constitutively and widelyexpressed on the surface of many cell types, including APCs (Foy et al.,Ann Rev. Immunol., 1996, 14, 591).

Signaling through CD40 by CD154 initiates a cascade of events thatresult in the activation of the CD40 receptor-bearing cells and optimalCD4+ T cell priming. More specifically, the cognate interaction betweenCD154 and CD40 promotes the differentiation of B cells into antibodysecreting cells and memory B cells (Burkly, In Adv. Exp. Med. Bio., Vol.489., D. M. Monroe, U. Hedner, M. R. Hoffman, C. Negrier, G. F. Savidge,and G. C. I. White, eds. Kluwer Academic/Plenum Publishers, 2001, p.135). Additionally, the CD154-CD40 interaction promotes cell-mediatedimmunity through the activation of macrophages and dendritic cells andthe generation of natural killer cells and cytotoxic T lymphocytes(Burkly, supra).

The CD40-CD154 interaction has been shown to be important in severalexperimentally induced autoimmune diseases, such as collagen-inducedarthritis, experimental allergic encephalomyelitis (“EAE”), oophoritis,colitis, drug-induced lupus nephritis. Specifically, it has been shownthat disease induction in all of these models can be blocked with CD154antagonists at the time of antigen administration (Burkly, supra).

The blockade of disease using anti-CD154 antagonists has also been seenin animal models of spontaneous autoimmune disease, includinginsulin-dependent diabetes and lupus nephritis, as well as ingraft-vs-host disease, transplant, pulmonary fibrosis, andatherosclerosis disease models (Burkly, supra).

Although glycosylated anti-CD154 antibodies have proven useful for theprevention and treatment of several immune response-related diseases, insome subjects, therapies using them are sometimes complicated bythromboembolitic activity. Although the mechanism of this side effect isunknown, it could involve the colligation by the anti-CD154 antibody, oraggregates thereof, of FcgRIIa and CD154 on platelets, leading toinappropriate platelet activation. Binding to other Fcγ receptors andcomplement could also potentiate this effect. Thus, forms of anti-CD154antibodies that do not bind to effector receptors may be safer and/ormore effective for therapeutic use.

The mechanism by which anti-CD154 antibodies inhibit immune function maybe more complex than simple binding to CD154 to block interactions withCD40 and, in fact, may include contributions by effector pathways. Forexample, antibody-antigen binding may induce deletion of activated Tcells through Fc domain binding to Fcγ receptors or complementcomponents. Alternatively, binding of the antibody to CD154 may beenhanced by the formation of a cell surface scaffold of the antibody onFcγ receptor-bearing cells. In addition, access of the antibody to itssite of action may be promoted by Fcγ receptor binding interactions.

The pivotal role of CD154 in regulating the function of both the humoraland cell-mediated immune response has provoked great interest in the useof inhibitors of this pathway for therapeutic immunomodulation (U.S.Pat. No. 5,474,771). As such, anti-CD154 antibodies have been shown tobe beneficial in a wide variety of models of immune response to othertherapeutic proteins or gene therapy, allergens, autoimmunity andtransplantation (U.S. Pat. No. 5,474,771; Burkly, supra).

Accordingly, there remains a need for antibodies that do not provoke astrong immune response but yet bind strongly to their antigens andmethods for identifying such antibodies, in particular, improved CD154antibodies.

SUMMARY OF THE INVENTION

The present invention provides peptides comprising an amino acidsequence at least 80% identical to SEQ ID NO:3, or a fragment thereof,wherein the amino acid at each of positions 24, 26, 27, 28, 30, 31, 32,33, 34, 35, 36, 38, 54, 57, 58, 59, 60, 93, 95, 96, 97, 98, 100, and101, independently, is any naturally occurring amino acid or anynon-naturally occurring amino acid, wherein the peptide does not consistof SEQ ID NO:1, and wherein the fragment comprises at least one ofpositions 24, 26, 27, 28, 30, 31, 32, 33, 34, 35, 36, 38, 54, 57, 58,59, 60, 93, 95, 96, 97, 98, 100, and 101, and when complexed with thewild type 5c8 heavy chain, the peptide or fragment thereof can bind toCD154.

In some embodiments, the amino acid at position 24 is selected from thegroup consisting of Arg, Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr,Trp, Tyr, Ala, Ile, Leu, Pro, Val, Asp, Glu, and Lys; the amino acid atposition 26 is selected from the group consisting of Ser, Asn, Cys, Gln,Gly, His, Met, Phe, Thr, Trp, Tyr, Ala, Ile, Leu, Pro, Val, Asp, andGlu; the amino acid at position 27 is selected from the group consistingof Gln, Asn, Cys, Gly, His, Met, Phe, Ser, Thr, Trp, Tyr, Asp, Glu, Arg,and Lys; the amino acid at position 28 is selected from the groupconsisting of Arg, Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp,Tyr, Asp, Glu, Ala, Ile, Leu, Pro, and Val; the amino acid at position30 is selected from the group consisting of Ser, Asn, Cys, Gln, Gly,His, Met, Phe, Thr, Trp, Tyr, Asp, Glu, Arg, and Lys; the amino acid atposition 31 is selected from the group consisting of Ser, Asn, Cys, Gln,Gly, His, Met, Phe, Thr, Trp, Tyr, Ala, Ile, Leu, Pro, Val, Arg, andLys; the amino acid at position 32 is selected from the group consistingof Ser, Asn, Cys, Gln, Gly, His, Met, Phe, Thr, Trp, Tyr, Arg, Ala, Ile,Leu, and Lys; the amino acid at position 33 is selected from the groupconsisting of Thr, Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Trp, Tyr,Asp, Glu, Arg, Ala, Val, and Lys; the amino acid at position 34 isselected from the group consisting of Tyr, Asn, Cys, Gln, Gly, His, Met,Phe, Ser, Thr, Trp Ala, Ile, Leu, Pro, Val, Asp, Glu, Arg, and Lys; theamino acid at position 35 is selected from the group consisting of Ser,Asn, Cys, Gln, Gly, His, Met, Phe, Thr, Trp, and Tyr; the amino acid atposition 36 is selected from the group consisting of Tyr, Asn, Cys, Gln,Gly, His, Met, Phe, Ser, Thr, Trp Asp, Ala, Leu, and Glu; the amino acidat position 38 is selected from the group consisting of His, Asn, Cys,Gln, Gly, Met, Phe, Ser, Thr, Trp, Tyr, Ala, Ile, Leu, Pro, Val, Asp,Glu, Arg, and Lys; the amino acid at position 54 is selected from thegroup consisting of Tyr, Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr,Trp Ala, Ile, Leu, Pro, Val, Asp, and Glu; the amino acid at position 57is selected from the group consisting of Asn, Cys, Gln, Gly, His, Met,Phe, Ser, Thr, Trp, Tyr, Asp, and Glu; the amino acid at position 58 isselected from the group consisting of Leu, Asn, Cys, Gln, Gly, His, Met,Phe, Ser, Thr, Trp Asp, and Glu; the amino acid at position 59 isselected from the group consisting of Glu, Asn, Cys, Gln, Gly, His, Met,Phe, Ser, Thr, Trp, Tyr, and Asp; the amino acid at position 60 isselected from the group consisting of Ser, Asp, and Glu; the amino acidat position 93 is selected from the group consisting of Gln, Asn, Cys,Gly, His, Met, Phe, Ser, Thr, Trp, Tyr, Ala, Ile, Leu, Pro, Val, Asp,Glu, Arg, and Lys; the amino acid at position 95 is selected from thegroup consisting of Ser, Asn, Cys, Gln, Gly, His, Met, Phe, Thr, Trp,Tyr, Asp, and Glu; the amino acid at position 96 is selected from thegroup consisting of Trp, Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr,Tyr, Asp, Glu, Arg, and Lys; the amino acid at position 97 is selectedfrom the group consisting of Glu and Asp; the amino acid at position 98is selected from the group consisting of Ile, Asn, Cys, Gln, Gly, His,Met, Phe, Ser, Thr, Trp, Tyr, Asp, Ala, Leu, Pro, Val, and Glu; theamino acid at position 100 is selected from the group consisting of Pro,Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, Tyr, Asp, and Glu; andthe amino acid at position 101 is selected from the group consisting ofThr, Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Trp, Tyr, Ala, Ile, Leu,Pro, Val, Asp, and Glu.

In some embodiments, the amino acid at position 26 is selected from thegroup consisting of Ser and Asp; the amino acid at position 27 isselected from the group consisting of Gln and Glu; the amino acid atposition 28 is selected from the group consisting of Arg and Glu; theamino acid at position 31 is selected from the group consisting of Ser,Ala, His, Asn, Thr, Val, and Trp; the amino acid at position 32 isselected from the group consisting of Ser, Ala, Phe, Ile, Leu, Met, andTrp; the amino acid at position 33 is selected from the group consistingof Thr, Ala, Phe, Met, Val, Trp, Asp, Arg, Tyr, and Gln; the amino acidat position 34 is selected from the group consisting of Tyr, Ala, Asp,Glu, Phe, Ile, Lys, Leu, Met, Arg, Val, and Trp; the amino acid atposition 36 is selected from the group consisting of Tyr, Ala, Phe, Leu,and Trp; the amino acid at position 54 is selected from the groupconsisting of Tyr and Glu; the amino acid at position 96 is selectedfrom the group consisting of Trp, Asp, Glu, His, Arg, Ser, and Thr; andthe amino acid at position 98 is selected from the group consisting ofIle, Ala, Phe, His, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, andTyr.

In some embodiments, the amino acid at position 26 is selected from thegroup consisting of Ser, Asp and Glu; the amino acid at position 27 isselected from the group consisting of Gln, Asp, and Glu; the amino acidat position 31 is selected from the group consisting of Ser, Asn, Cys,Gln, Gly, Thr, and Tyr; the amino acid at position 32 is selected fromthe group consisting of Ser, Ala, Ile, Leu, Met, Phe, Pro, Val, and Trp;the amino acid at position 33 is selected from the group consisting ofThr, Asn, Cys, Gln, Gly, Ser, and Tyr; the amino acid at position 98 isselected from the group consisting of Ile, Asn, Cys, Gln, Gly, Ser, Thr,and Tyr; the amino acid at position 24 and position 28 is Arg; the aminoacid at position 30, position 35, position 60, and position 95 is Ser;the amino acid at position 34, position 36, and position 54 is Tyr; theamino acid at position 38 is His; the amino acid at position 57 is Asn;the amino acid at position 58 is Leu; the amino acid at position 59 andposition 97 is Glu; the amino acid at position 93 is Gln; the amino acidat position 96 is Trp; the amino acid at position 100 is Pro; and theamino acid at position 101 is Thr.

In some embodiments, the amino acid at position is 26 Asp; the aminoacid at position 27 is Glu; the amino acid at position 28 is Glu; theamino acid at position 31 is Val; the amino acid at position 33 isselected from the group consisting of Asp and Arg; and the amino acid atposition 54 is Glu.

In some embodiments, the amino acid at position 31 is selected from thegroup consisting of His and Asn; the amino acid at position 32 isselected from the group consisting of Trp and Phe; the amino acid atposition 33 is selected from the group consisting of Trp, Tyr, and Gln;the amino acid at position 36 is selected from the group consisting ofLeu and Trp; the amino acid at position 96 is His; and the amino acid atposition 98 is selected from the group consisting of Phe and Gln.

In some embodiments, the amino acid at position 26 is selected from thegroup consisting of Ser and Asp; the amino acid at position 27 isselected from the group consisting of Gln and Glu; the amino acid atposition 31 is selected from the group consisting of Ser and Asn; theamino acid at position 32 is selected from the group consisting of Serand Phe; the amino acid at position 33 is selected from the groupconsisting of Thr, Gln, and Tyr; the amino acid at position 98 isselected from the group consisting of Ile and Gln; the amino acid atposition 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Gln; theamino acid at position 31 and position 32 is Ser; the amino acid atposition 33 is Thr; the amino acid at position 98 is Ile; the amino acidat position 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26, position 31, and position 32 is Ser; the amino acidat position 27 is Glu; the amino acid at position 33 is Thr; the aminoacid at position 98 is Ile; the amino acid at position 24 and position28 is Arg; the amino acid at position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 34, position 36, andposition 54 is Tyr; the amino acid at position 38 is His; the amino acidat position 57 is Asn; the amino acid at position 58 is Leu; the aminoacid at position 59 and position 97 is Glu; the amino acid at position93 is Gln; the amino acid at position 96 is Trp; the amino acid atposition 100 is Pro; and the amino acid at position 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 and position 32 is Ser; the amino acid at position27 is Gln; the amino acid at position 31 is Asn; the amino acid atposition 33 is Thr; the amino acid at position 98 is Ile; the amino acidat position 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 and position 31 is Ser; the amino acid at position27 is Gln; the amino acid at position 32 is Phe; the amino acid atposition 33 is Thr; the amino acid at position 98 is Ile; the amino acidat position 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26, position 31, and position 32 is Ser; the amino acidat position 27 and position 33 is Gln; the amino acid at position 98 isIle; the amino acid at position 24 and position 28 is Arg; the aminoacid at position 30, position 35, position 60, and position 95 is Ser;the amino acid at position 34, position 36, and position 54 is Tyr; theamino acid at position 38 is His; the amino acid at position 57 is Asn;the amino acid at position 58 is Leu; the amino acid at position 59 andposition 97 is Glu; the amino acid at position 93 is Gln; the amino acidat position 96 is Trp; the amino acid at position 100 is Pro; and theamino acid at position 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26, position 31, and position 32 is Ser; the amino acidat position 27 is Gln; the amino acid at position 33 is Tyr; the aminoacid at position 98 is Ile; the amino acid at position 24 and position28 is Arg; the amino acid at position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 34, position 36, andposition 54 is Tyr; the amino acid at position 38 is His; the amino acidat position 57 is Asn; the amino acid at position 58 is Leu; the aminoacid at position 59 and position 97 is Glu; the amino acid at position93 is Gln; the amino acid at position 96 is Trp; the amino acid atposition 100 is Pro; and the amino acid at position 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26, position 31, and position 32 is Ser; the amino acidat position 27 and position 98 is Gln; the amino acid at position 33 isThr; the amino acid at position 24 and position 28 is Arg; the aminoacid at position 30, position 35, position 60, and position 95 is Ser;the amino acid at position 34, position 36, and position 54 is Tyr; theamino acid at position 38 is His; the amino acid at position 57 is Asn;the amino acid at position 58 is Leu; the amino acid at position 59 andposition 97 is Glu; the amino acid at position 93 is Gln; the amino acidat position 96 is Trp; the amino acid at position 100 is Pro; and theamino acid at position 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Glu; theamino acid at position 31 and position 32 is Ser; the amino acid atposition 33 is Thr; the amino acid at position 98 is Ile; the amino acidat position 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Glu; theamino acid at position 31 is Asn; the amino acid at position 32 is Ser;the amino acid at position 33 is Thr; the amino acid at position 98 isIle; the amino acid at position 24 and position 28 is Arg; the aminoacid at position 30, position 35, position 60, and position 95 is Ser;the amino acid at position 34, position 36, and position 54 is Tyr; theamino acid at position 38 is His; the amino acid at position 57 is Asn;the amino acid at position 58 is Leu; the amino acid at position 59 andposition 97 is Glu; the amino acid at position 93 is Gln; the amino acidat position 96 is Trp; the amino acid at position 100 is Pro; and theamino acid at position 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Glu; theamino acid at position 31 is Ser; the amino acid at position 32 is Phe;the amino acid at position 33 is Thr; the amino acid at position 98 isIle; the amino acid at position 24 and position 28 is Arg; the aminoacid at position 30, position 35, position 60, and position 95 is Ser;the amino acid at position 34, position 36, and position 54 is Tyr; theamino acid at position 38 is His; the amino acid at position 57 is Asn;the amino acid at position 58 is Leu; the amino acid at position 59 andposition 97 is Glu; the amino acid at position 93 is Gln; the amino acidat position 96 is Trp; the amino acid at position 100 is Pro; and theamino acid at position 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Glu; theamino acid at position 31 and position 32 is Ser; the amino acid atposition 33 is Gln; the amino acid at position 98 is Ile; the amino acidat position 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Glu; theamino acid at position 31 and position 32 is Ser; the amino acid atposition 33 is Tyr; the amino acid at position 98 is Ile; the amino acidat position 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Glu; theamino acid at position 31 and position 32 is Ser; the amino acid atposition 33 is Thr; the amino acid at position 98 is Gln; the amino acidat position 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 27 and position 93 is Gln; the amino acid at position26, position 31, position 32, position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 33 and position 101 isThr; the amino acid at position 98 is Ile; the amino acid at position 24and position 28 is Arg; the amino acid at position 34 and position 54 isTyr; the amino acid at position 38 is His; the amino acid at position 57is Asn; the amino acid at position 58 is Leu; the amino acid at position59 and position 97 is Glu; the amino acid at position 96 and position 36is Trp; and the amino acid at position 100 is Pro.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 27 and position 93 is Gln; the amino acid at position26, position 31, position 32, position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 101 is Thr; the aminoacid at position 98 is Ile; the amino acid at position 24 and position28 is Arg; the amino acid at position 34, position 36, and position 54is Tyr; the amino acid at position 38 is His; the amino acid at position57 is Asn; the amino acid at position 58 is Leu; the amino acid atposition 59 and position 97 is Glu; the amino acid at position 96 andposition 33 is Trp; and the amino acid at position 100 is Pro.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 27 and position 93 is Gln; the amino acid at position26, position 32, position 30, position 35, position 60, and position 95is Ser; the amino acid at position 33 and position 101 is Thr; the aminoacid at position 98 is Ile; the amino acid at position 24 and position28 is Arg; the amino acid at position 34, position 36, and position 54is Tyr; the amino acid at position 38 is His; the amino acid at position57 is Asn; the amino acid at position 58 is Leu; the amino acid atposition 59 and position 97 is Glu; the amino acid at position 96 isTrp; the amino acid at position 31 is Val; and the amino acid atposition 100 is Pro.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 27 and position 93 is Gln; the amino acid at position26, position 31, position 32, position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 101 is Thr; the aminoacid at position 98 is Ile; the amino acid at position 24 and position28 is Arg; the amino acid at position 34, position 36, and position 54is Tyr; the amino acid at position 38 is His; the amino acid at position57 is Asn; the amino acid at position 58 is Leu; the amino acid atposition 59 and position 97 is Glu; the amino acid at position 96 isTrp; the amino acid at position 33 is Asp; and the amino acid atposition 100 is Pro.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 27 and position 93 is Gln; the amino acid at position26, position 31, position 32, position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 33 and position 101 isThr; the amino acid at position 98 is Ile; the amino acid at position 24and position 28 is Arg; the amino acid at position 34 and position 36 isTyr; the amino acid at position 38 is His; the amino acid at position 57is Asn; the amino acid at position 58 is Leu; the amino acid at position54, position 59 and position 97 is Glu; the amino acid at position 96 isTrp; and the amino acid at position 100 is Pro.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 27 and position 93 is Gln; the amino acid at position26, position 31, position 32, position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 33 and position 101 isThr; the amino acid at position 98 is Ile; the amino acid at position 24and position 28 is Arg; the amino acid at position 34, position 36, andposition 54 is Tyr; the amino acid at position 38 is His; the amino acidat position 57 is Asn; the amino acid at position 58 is Leu; the aminoacid at position 59 and position 97 is Glu; the amino acid at position96 is Trp; and the amino acid at position 100 is Pro.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Glu; theamino acid at position 31 and position 32 is Ser; the amino acid atposition 98 is Ile; the amino acid at position 24 and position 28 isArg; the amino acid at position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 34, position 36, andposition 54 is Tyr; the amino acid at position 38 is His; the amino acidat position 57 is Asn; the amino acid at position 58 is Leu; the aminoacid at position 59 and position 97 is Glu; the amino acid at position93 is Gln; the amino acid at position 33 and position 96 is Trp; theamino acid at position 100 is Pro; and the amino acid at position 101 isThr.

The present invention also provides peptides that comprise an amino acidsequence at least 80% identical to SEQ ID NO:6, or a fragment thereof,wherein the amino acid at each of positions 28, 30, 31, 32, 33, 35, 50,52, 53, 54, 55, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 99, 100, 101,102, 103, 104, 105, 106, and 107, independently, is any naturallyoccurring amino acid or any non-naturally occurring amino acid, whereinthe peptide does not consist of SEQ ID NO:4, and wherein the fragmentcomprises at least one of positions 28, 30, 31, 32, 33, 35, 50, 52, 53,54, 55, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 99, 100, 101, 102, 103,104, 105, 106, and 107, and when complexed with the wild type 5c8 lightchain, the peptide or fragment thereof can bind to CD154.

In some embodiments, the amino acid at position 28 is selected from thegroup consisting of Ile, Asp, Glu, Arg, Lys, Asn, Cys, Gln, Gly, His,Met, Phe, Ser, Thr, Trp and Trp; the amino acid at position 30 isselected from the group consisting of Thr, Asp, Glu, Arg, Lys, Asn, Cys,Gln, Gly, His, Met, Phe, Ser, Trp, Tyr, Ala, Ile, Leu, Pro, and Val; theamino acid at position 31 is selected from the group consisting of Ser,Arg, His, Lys, Gln, and Trp; the amino acid at position 32 is selectedfrom the group consisting of Tyr, Asp, Glu, Arg, Lys, Asn, Cys, Gln,Gly, His, Met, Phe, Ser, Trp, Ala, Ile, Leu, Pro, and Val; the aminoacid at position 33 is selected from the group consisting of Tyr, Asp,Glu, Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp Ala, Ile, Leu,Pro, and Val; the amino acid at position 35 is selected from the groupconsisting of Tyr, Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp Asp,and Glu; the amino acid at position 50 is selected from the groupconsisting of Glu and Asp; the amino acid at position 52 is selectedfrom the group consisting of Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Trp, Tyr, Arg, Ala, Leu, Val, and Lys; the amino acid at position53 is selected from the group consisting of Pro, Asn, Cys, Gln, Gly,His, Met, Phe, Ser, Thr, Trp, Tyr, Asp, and Glu; the amino acid atposition 54 is selected from the group consisting of Ser, Asn, Cys, Gln,Gly, His, Met, Phe, Thr, Trp, Tyr, Arg, Lys, and Glu; the amino acid atposition 55 is selected from the group consisting of Asn, Glu, Lys, Gln,Ser, Thr, Met, and Val; the amino acid at position 57 is selected fromthe group consisting of Asp, Glu, Phe, and Leu; the amino acid atposition 58 is selected from the group consisting of Thr, Asn, Cys, Gln,Gly, His, Met, Phe, Ser, Trp, Tyr, Ala, Ile, Leu, Pro, and Val; theamino acid at position 59 is selected from the group consisting of Asn,Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, Tyr, Ala, Ile, Leu, Pro,Val, Asp, and Glu; the amino acid at position 60 is selected from thegroup consisting of Phe, Asp, Asn, Cys, Gln, Gly, His, Met, Ser, Thr,Trp, Tyr, Glu, Arg, Lys, Ala, Ile, Leu, Pro, and Val; the amino acid atposition 61 is selected from the group consisting of Asn, Asp, and Glu;the amino acid at position 62 is selected from the group consisting ofGlu and Asp; the amino acid at position 63 is selected from the groupconsisting of Lys, Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp,Tyr, Arg, Glu, Asp, Ala, Ile, Leu, Pro, and Val; the amino acid atposition 64 is selected from the group consisting of Phe, Asn, Cys, Gln,Gly, His, Met, Ser, Thr, Trp, Tyr, Asp, Glu, Ala, Ile, Leu, Pro, andVal; the amino acid at position 65 is selected from the group consistingof Lys, Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, Tyr, Arg, Asp,Glu, Ala, Ile, Leu, Pro, and Val; the amino acid at position 66 isselected from the group consisting of Ser, Asp, and Glu; the amino acidat position 99 is selected from the group consisting of Ser, Asp, Glu,and Ala; the amino acid at position 100 is selected from the groupconsisting of Asp and Glu; the amino acid at position 101 is selectedfrom the group consisting of Gly, Phe, and Leu; the amino acid atposition 102 is selected from the group consisting of Arg, Asn, Cys,Gln, Gly, His, Met, Phe, Ser, Thr, Trp, Tyr, and Lys; the amino acid atposition 103 is selected from the group consisting of Asn, Cys, Gln,Gly, His, Met, Phe, Ser, Thr, Trp, Tyr, Asp, Glu, Ala, Ile, Lys, Arg,and Val; the amino acid at position 104 is selected from the groupconsisting of Asp and Glu; the amino acid at position 105 is selectedfrom the group consisting of Met, Asp, Glu, Arg, His, Lys, Asn, Cys,Gln, Gly, Phe, Ser, Thr, Trp, and Tyr; the amino acid at position 106 isselected from the group consisting of Asp and Glu; and the amino acid atposition 107 is selected from the group consisting of Ser, Asn, Cys,Gln, Gly, His, Met, Phe, Thr, Trp, Tyr, Asp, Glu, Ala, Ile, Leu, Pro,and Val.

In some embodiments, the amino acid at position 28 is selected from thegroup consisting of Ile, His, Asn, Gln, Ser, Thr, and Glu; the aminoacid at position 30 is selected from the group consisting of Thr, His,Asn, Gln, Ser, Tyr, and Arg; the amino acid at position 31 is selectedfrom the group consisting of Ser, Gln, and Trp; the amino acid atposition 33 is selected from the group consisting of Tyr, Ala, Pro, Ser,Thr, Val, and Trp; the amino acid at position 52 is selected from thegroup consisting of Asn, Ala, Phe, His, Leu, Met, Ser, Thr, Val, andTrp; the amino acid at position 54 is selected from the group consistingof Ser, Glu, His, Lys, Asn, Gln, Arg, Thr, Trp, Tyr, and Phe; the aminoacid at position 55 is selected from the group consisting of Asn, Glu,Lys, Gln, Ser, Thr, Met, and Val; the amino acid at position 57 isselected from the group consisting of Asp, Phe, and Leu; the amino acidat position 59 is selected from the group consisting of Asn, Ala, Phe,Leu, Met, Pro, Val, Trp, Asp, and Tyr; the amino acid at position 99 isselected from the group consisting of Ser and Ala; the amino acid atposition 101 is selected from the group consisting of Gly, Phe, and Leu;and the amino acid at position 103 is selected from the group consistingof Asn, Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Arg, Ser, Thr, Val, andTyr.

In some embodiments, the amino acid at position 31 and position 54 is,independently, selected from the group consisting of Ser, Ala, Ile, Leu,Met, Phe, Pro, Val, Trp, Asn, Cys, Gln, Gly, Ser, Thr, and Tyr; theamino acid at position 57 is selected from the group consisting of Asp,Ala, Ile, Leu, Met, Phe, Pro, Val, and Trp; the amino acid at position101 is selected from the group consisting of Gly, Ala, Ile, Leu, Met,Phe, Pro, Val, and Trp; the amino acid at position 103 is selected fromthe group consisting of Asn, Cys, Gln, Gly, Ser, Thr, and Tyr; the aminoacid at position 28 is Ile; the amino acid at position 30 and position58 is Thr; the amino acid at position 32, position 33, and position 35is Tyr; the amino acid at position 50 and position 62 is Glu; the aminoacid at position 52, position 55, position 59, and position 61 is Asn;the amino acid at position 53 is Pro; the amino acid at position 60 andposition 64 is Phe; the amino acid at position 63 and position 65 isLys; the amino acid at position 66, position 99, and position 107 isSer; the amino acid at position 100, position 104, and position 106 isAsp; the amino acid at position 102 is Arg; and the amino acid atposition 105 is Met.

In some embodiments, the amino acid at position 31 is selected from thegroup consisting of Ser, Trp, and Gln; the amino acid at position 54 isselected from the group consisting of Ser, Phe, and Gln; the amino acidat position 57 is selected from the group consisting of Asp, Leu, andPhe; the amino acid at position 101 is selected from the groupconsisting of Gly, Leu, and Phe; the amino acid at position 103 isselected from the group consisting of Asn and Tyr; the amino acid atposition 28 is Ile; the amino acid at position 30 and position 58 isThr; the amino acid at position 32, position 33, and position 35 is Tyr;the amino acid at position 50 and position 62 is Glu; the amino acid atposition 52, position 55, position 59, and position 61 is Asn; the aminoacid at position 53 is Pro; the amino acid at position 60 and position64 is Phe; the amino acid at position 63 and position 65 is Lys; theamino acid at position 66, position 99, and position 107 is Ser; theamino acid at position 100, position 104, and position 106 is Asp; theamino acid at position 102 is Arg; and the amino acid at position 105 isMet.

In some embodiments, the amino acid at position 28 is Glu; the aminoacid at position 33 is Phe; the amino acid at position 54 is Thr; andthe amino acid at position 59 is selected from the group consisting ofAsp and Leu.

In some embodiments, the amino acid at position 30 is selected from thegroup consisting of His and Arg; the amino acid at position 31 isselected from the group consisting of Gln and Trp; the amino acid atposition 33 is selected from the group consisting of Trp, Val, and Pro;the amino acid at position 52 is selected from the group consisting ofMet and Trp; the amino acid at position 54 is selected from the groupconsisting of Asn, Phe, and Gln; the amino acid at position 55 isselected from the group consisting of Met, Lys, and Val; the amino acidat position 57 is selected from the group consisting of Phe and Leu; theamino acid at position 59 is selected from the group consisting of Pheand Tyr; the amino acid at position 101 is selected from the groupconsisting of Phe and Leu; and the amino acid at position 103 isselected from the group consisting of His and Tyr.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 is Trp; the amino acid at position 54 is Ser; theamino acid at position 57 is Asp; the amino acid at position 101 is Gly;the amino acid at position 103 is Asn; the amino acid at position 28 isIle; the amino acid at position 30 and position 58 is Thr; the aminoacid at position 32, position 33, and position 35 is Tyr; the amino acidat position 50 and position 62 is Glu; the amino acid at position 52,position 55, position 59, and position 61 is Asn; the amino acid atposition 53 is Pro; the amino acid at position 60 and position 64 isPhe; the amino acid at position 63 and position 65 is Lys; the aminoacid at position 66, position 99, and position 107 is Ser; the aminoacid at position 100, position 104, and position 106 is Asp; the aminoacid at position 102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 is Gln; the amino acid at position 54 is Ser; theamino acid at position 57 is Asp; the amino acid at position 101 is Gly;the amino acid at position 103 is Asn; the amino acid at position 28 isIle; the amino acid at position 30 and position 58 is Thr; the aminoacid at position 32, position 33, and position 35 is Tyr; the amino acidat position 50 and position 62 is Glu; the amino acid at position 52,position 55, position 59, and position 61 is Asn; the amino acid atposition 53 is Pro; the amino acid at position 60 and position 64 isPhe; the amino acid at position 63 and position 65 is Lys; the aminoacid at position 66, position 99, and position 107 is Ser; the aminoacid at position 100, position 104, and position 106 is Asp; the aminoacid at position 102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 is Ser; the amino acid at position 54 is Phe; theamino acid at position 57 is Asp; the amino acid at position 101 is Gly;the amino acid at position 103 is Asn; the amino acid at position 28 isIle; the amino acid at position 30 and position 58 is Thr; the aminoacid at position 32, position 33, and position 35 is Tyr; the amino acidat position 50 and position 62 is Glu; the amino acid at position 52,position 55, position 59, and position 61 is Asn; the amino acid atposition 53 is Pro; the amino acid at position 60 and position 64 isPhe; the amino acid at position 63 and position 65 is Lys; the aminoacid at position 66, position 99, and position 107 is Ser; the aminoacid at position 100, position 104, and position 106 is Asp; the aminoacid at position 102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 is Ser; the amino acid at position 54 is Gln; theamino acid at position 57 is Asp; the amino acid at position 101 is Gly;the amino acid at position 103 is Asn; the amino acid at position 28 isIle; the amino acid at position 30 and position 58 is Thr; the aminoacid at position 32, position 33, and position 35 is Tyr; the amino acidat position 50 and position 62 is Glu; the amino acid at position 52,position 55, position 59, and position 61 is Asn; the amino acid atposition 53 is Pro; the amino acid at position 60 and position 64 isPhe; the amino acid at position 63 and position 65 is Lys; the aminoacid at position 66, position 99, and position 107 is Ser; the aminoacid at position 100, position 104, and position 106 is Asp; the aminoacid at position 102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 and position 54 is Ser; the amino acid at position57 is Phe; the amino acid at position 101 is Gly; the amino acid atposition 103 is Asn; the amino acid at position 28 is Ile; the aminoacid at position 30 and position 58 is Thr; the amino acid at position32, position 33, and position 35 is Tyr; the amino acid at position 50and position 62 is Glu; the amino acid at position 52, position 55,position 59, and position 61 is Asn; the amino acid at position 53 isPro; the amino acid at position 60 and position 64 is Phe; the aminoacid at position 63 and position 65 is Lys; the amino acid at position66, position 99, and position 107 is Ser; the amino acid at position100, position 104, and position 106 is Asp; the amino acid at position102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 and position 54 is Ser; the amino acid at position57 is Leu; the amino acid at position 101 is Gly; the amino acid atposition 103 is Asn; the amino acid at position 28 is Ile; the aminoacid at position 30 and position 58 is Thr; the amino acid at position32, position 33, and position 35 is Tyr; the amino acid at position 50and position 62 is Glu; the amino acid at position 52, position 55,position 59, and position 61 is Asn; the amino acid at position 53 isPro; the amino acid at position 60 and position 64 is Phe; the aminoacid at position 63 and position 65 is Lys; the amino acid at position66, position 99, and position 107 is Ser; the amino acid at position100, position 104, and position 106 is Asp; the amino acid at position102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 and position 54 is Ser; the amino acid at position57 is Asp; the amino acid at position 101 is Phe; the amino acid atposition 103 is Asn; the amino acid at position 28 is Ile; the aminoacid at position 30 and position 58 is Thr; the amino acid at position32, position 33, and position 35 is Tyr; the amino acid at position 50and position 62 is Glu; the amino acid at position 52, position 55,position 59, and position 61 is Asn; the amino acid at position 53 isPro; the amino acid at position 60 and position 64 is Phe; the aminoacid at position 63 and position 65 is Lys; the amino acid at position66, position 99, and position 107 is Ser; the amino acid at position100, position 104, and position 106 is Asp; the amino acid at position102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 and position 54 is Ser; the amino acid at position57 is Asp; the amino acid at position 101 is Leu; the amino acid atposition 103 is Asn; the amino acid at position 28 is Ile; the aminoacid at position 30 and position 58 is Thr; the amino acid at position32, position 33, and position 35 is Tyr; the amino acid at position 50and position 62 is Glu; the amino acid at position 52, position 55,position 59, and position 61 is Asn; the amino acid at position 53 isPro; the amino acid at position 60 and position 64 is Phe; the aminoacid at position 63 and position 65 is Lys; the amino acid at position66, position 99, and position 107 is Ser; the amino acid at position100, position 104, and position 106 is Asp; the amino acid at position102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 and position 54 is Ser; the amino acid at position57 is Asp; the amino acid at position 101 is Gly; the amino acid atposition 103 is Tyr; the amino acid at position 28 is Ile; the aminoacid at position 30 and position 58 is Thr; the amino acid at position32, position 33, and position 35 is Tyr; the amino acid at position 50and position 62 is Glu; the amino acid at position 52, position 55,position 59, and position 61 is Asn; the amino acid at position 53 isPro; the amino acid at position 60 and position 64 is Phe; the aminoacid at position 63 and position 65 is Lys; the amino acid at position66, position 99, and position 107 is Ser; the amino acid at position100, position 104, and position 106 is Asp; the amino acid at position102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31, position 54, position 66, position 99, and position107 is Ser; the amino acid at position 57, position 100, position 104,and position 106 is Asp; the amino acid at position 101 is Gly; theamino acid at position 103, position 52, position 55, position 59, andposition 61 is Asn; the amino acid at position 28 is Ile; the amino acidat position 58 is Thr; the amino acid at position 32, position 33, andposition 35 is Tyr; the amino acid at position 50 and position 62 isGlu; the amino acid at position 53 is Pro; the amino acid at position 60and position 64 is Phe; the amino acid at position 63 and position 65 isLys; the amino acid at position 102 is Arg; the amino acid at position30 is His; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31, position 54, position 66, position 99, and position107 is Ser; the amino acid at position 57, position 100, position 104,and position 106 is Asp; the amino acid at position 101 is Gly; theamino acid at position 103, position 52, position 55, position 59, andposition 61 is Asn; the amino acid at position 28 is Ile; the amino acidat position 30 and position 58 is Thr; the amino acid at position 32,and position 35 is Tyr; the amino acid at position 50 and position 62 isGlu; the amino acid at position 53 is Pro; the amino acid at position 60and position 64 is Phe; the amino acid at position 63 and position 65 isLys; the amino acid at position 102 is Arg; the amino acid at position33 is Trp; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31, position 66, position 99, and position 107 is Ser;the amino acid at position 57, position 100, position 104, and position106 is Asp; the amino acid at position 101 is Gly; the amino acid atposition 103, position 52, position 54, position 55, position 59, andposition 61 is Asn; the amino acid at position 28 is Ile; the amino acidat position 30 and position 58 is Thr; the amino acid at position 32,position 33, and position 35 is Tyr; the amino acid at position 50 andposition 62 is Glu; the amino acid at position 53 is Pro; the amino acidat position 60 and position 64 is Phe; the amino acid at position 63 andposition 65 is Lys; the amino acid at position 102 is Arg; and the aminoacid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31, position 54, position 66, position 99, and position107 is Ser; the amino acid at position 57, position 100, position 104,and position 106 is Asp; the amino acid at position 101 is Gly; theamino acid at position 103, position 52, position 55, position 59, andposition 61 is Asn; the amino acid at position 28 is Ile; the amino acidat position 30 and position 58 is Thr; the amino acid at position 32,and position 35 is Tyr; the amino acid at position 50 and position 62 isGlu; the amino acid at position 53 is Pro; the amino acid at position33, position 60, and position 64 is Phe; the amino acid at position 63and position 65 is Lys; the amino acid at position 102 is Arg; and theamino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31, position 54, position 66, position 99, and position107 is Ser; the amino acid at position 57, position 59, position 100,position 104, and position 106 is Asp; the amino acid at position 101 isGly; the amino acid at position 103, position 52, position 55, andposition 61 is Asn; the amino acid at position 28 is Ile; the amino acidat position 30 and position 58 is Thr; the amino acid at position 32,position 33, and position 35 is Tyr; the amino acid at position 50 andposition 62 is Glu; the amino acid at position 53 is Pro; the amino acidat position 60 and position 64 is Phe; the amino acid at position 63 andposition 65 is Lys; the amino acid at position 102 is Arg; and the aminoacid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31, position 54, position 66, position 99, and position107 is Ser; the amino acid at position 57, position 100, position 104,and position 106 is Asp; the amino acid at position 101 is Gly; theamino acid at position 103, position 52, position 55, and position 61 isAsn; the amino acid at position 28 is Ile; the amino acid at position 30and position 58 is Thr; the amino acid at position 32, position 33, andposition 35 is Tyr; the amino acid at position 50 and position 62 isGlu; the amino acid at position 53 is Pro; the amino acid at position 60and position 64 is Phe; the amino acid at position 63 and position 65 isLys; the amino acid at position 102 is Arg; the amino acid at position59 is Leu; and the amino acid at position 105 is Met.

The present invention also provides compositions comprising any of thepeptide disclosed herein.

The present invention also provides nucleic acid molecules encoding anyof the peptides disclosed herein.

The present invention also provides vectors comprising any of thenucleic acid molecules disclosed herein.

The present invention also provides compositions comprising any of thenucleic acid molecules or vectors disclosed herein.

The present invention also provides host cells comprising any of thenucleic acid molecules or vectors disclosed herein.

The present invention also provides antibodies, or fragments thereof,comprising any of the peptides disclosed herein, wherein the antibodiesor fragments thereof can bind to human CD154.

In some embodiments, the antibody fragment is selected from the groupconsisting of a single chain antibody (scFv), a F(ab′)₂ fragment, a Fabfragment, and an Fd fragment.

In some embodiments, the antibody, or fragment thereof, is labeled witha detectable marker, such as, a marker selected from the groupconsisting of radioactive isotope, enzyme, fluorochrome, colloidal gold,dye, and biotin.

In some embodiments, the antibody, or fragment thereof, is conjugated toa therapeutic agent, such as, one selected from the group consisting ofa radioisotope, toxin, toxoid, and chemotherapeutic agent; or isconjugated to a bead.

In some embodiments, the antibody, or fragment thereof, comprises atleast one high molecular-weight polymer, such as, one selected from thegroup consisting of polyethyleneimine and polylysine.

In some embodiments, the antibody, or fragment thereof, comprises atleast one amino acid that is selected from the group consisting ofPEGylated and glycosylated.

The present invention also provides compositions comprising any of theantibodies, or fragments thereof, disclosed herein.

The present invention also provides kits comprising any of theantibodies, or fragments thereof, disclosed herein.

The present invention also provides methods of treating or preventing aCD154-related human disease or disorder comprising administering to ahuman a therapeutically- or prophylactically-effective amount of anantibody, or fragment thereof, of any of claims 55 to 77, or acomposition of claim 53, such that the CD154-related human disease ordisorder is diminished or prevented.

In some embodiments, the human disease or disorder is inflammation, suchas, one selected from the group consisting of inflammation associatedwith arthritis, contact dermatitis, hyper-IgE syndrome, inflammatorybowel disease, allergic asthma, and idiopathic inflammatory disease.

In some embodiments, the arthritis is selected from the group consistingof rheumatoid arthritis, non-rheumatoid inflammatory arthritis,arthritis associated with Lyme disease, and inflammatory osteoarthritis.

In some embodiments, the idiopathic inflammatory disease is selectedfrom the group consisting of psoriasis and systemic lupus erythematosus.

In some embodiments, the disease or disorder is selected from the groupconsisting of Myasthenia gravis, Graves' disease, idiopathicthrombocytopenia purpura, hemolytic anemia, diabetes mellitus, Crohn'sdisease, multiple sclerosis, and drug-induced autoimmune diseases.

In some embodiments, the disorder is rejection by the subject of atransplanted organ, such as, one selected from the group consisting of atransplanted heart, kidney, liver, skin, pancreatic islet cells, andbone marrow.

In some embodiments, the disorder is selected from the group consistingof graft-vs-host disease, allergic responses, an autoimmune response,and fibrosis in a subject, such as, pulmonary fibrosis or fibroticdisease.

In some embodiments, the pulmonary fibrosis is selected from the groupconsisting of pulmonary fibrosis secondary to adult respiratory distresssyndrome, drug-induced pulmonary fibrosis, idiopathic pulmonaryfibrosis, and hypersensitivity pneumonitis; and wherein the fibroticdisease is selected from the group consisting of Hepatitis-C,Hepatitis-B, cirrhosis, cirrhosis of the liver secondary to a toxicinsult, cirrhosis of the liver secondary to drugs, cirrhosis of theliver secondary to a viral infection, and cirrhosis of the liversecondary to an autoimmune disease.

In some embodiments, the autoimmune response is selected from the groupconsisting of one derived from Reiter' syndrome, spondyloarthritis, Lymedisease, HIV infection, syphilis, and tuberculosis.

In some embodiments, the disease or disorder is gastrointestinaldisease, such as, one selected from the group consisting of esophagealdysmotility, inflammatory bowel disease, and scleroderma; or is avascular disease, wherein the vascular disease is selected from thegroup consisting of atherosclerosis and reperfusion injury.

In some embodiments, the disease or disorder is a T cell tumor cancer,such as, one selected from the group consisting of a T cell leukemia andlymphoma.

The present invention also provides peptides, and fragments thereof, foruse in treating a human disease or disorder associated with CD154 and inthe manufacture of a medicament for the treatment of a human disease ordisorder associated with CD154.

DESCRIPTION OF EMBODIMENTS

The present invention provides peptides, particularly light chain andheavy chain variable regions, and fragments thereof, and antibodies, andfragments thereof, comprising the same, wherein the peptides comprise atleast one amino acid substitution compared to wild type 5c8 antibody.

The term “antibody”, as used herein, includes monoclonal antibodies(including full length monoclonal antibodies), polyclonal antibodies,multispecific antibodies (for example, bispecific antibodies), chimericantibodies, CDR-grafted antibodies, humanized antibodies, humanantibodies, and the like, and fragments thereof.

The phrase “antibody fragment”, or the like, as used herein, includesfor example, an antibody light chain (V_(L)), an antibody heavy chain(V_(H)), a single chain antibody (scFv), a F(ab′)₂ fragment, a Fabfragment, a Fab′ fragment, an Fd fragment, an Fv fragment, and a singledomain antibody fragment (DAb).

The term “CDR”, as used herein, includes the complementarity determiningregions as described by, for example Kabat, Chothia, or MacCallum etal., (see, for example, Kabat et al., In “Sequences of Proteins ofImmunological Interest,” U.S. Department of Health and Human Services,1983; Chothia et al., J. Mol. Biol., 1987, 196, 901-917; and MacCallumet al., J. Mol. Biol., 1996, 262, 732-745).

The amino acid residue positions which typically encompass the CDRs asdescribed by each of the above cited references are set forth below forcomparison.

CDR Definitions Kabat Chothia MacCallum 5c8 VH CDR1 31-35 26-32 30-3526-35 VH CDR2 50-65 53-55 47-58 50-66 VH CDR3  95-102  96-101  93-101 99-107 VL CDR1 24-34 26-32 30-36 24-38 VL CDR2 50-56 50-52 46-55 54-60VL CDR3 89-97 91-96 89-96  93-101

The phrase “framework region”, as used herein, includes the antibodysequence that is between and separates the CDRs. Therefore, a variableregion framework is between about 100 to 120 amino acids in length butis intended to reference only those amino acids outside of the CDRs. Forthe specific example of a heavy chain variable region and for the CDRsas defined by Kabat et al., framework region 1 corresponds to the domainof the variable region encompassing amino acids 1 to 30; region 2corresponds to the domain of the variable region encompassing aminoacids 36 to 49; region 3 corresponds to the domain of the variableregion encompassing amino acids 66 to 94, and region 4 corresponds tothe domain of the variable region from amino acids 103 to the end of thevariable region. The framework regions for the light chain are similarlyseparated by each of the light claim variable region CDRs. Similarly,using the definition of CDRs by Chothia et al. or McCallum et al. theframework region boundaries are separated by the respective CDR terminias described above.

The phrase “variable region”, as used herein, includes the aminoterminal portion of an antibody which confers antigen binding onto themolecule and which is not the constant region. The term is intended toinclude functional fragments, for example, antigen-binding fragments,which maintain some or all of the binding function of the whole variableregion.

The phrase “5c8 antibody” refers to an antibody that binds CD154 and isclaimed and described in U.S. Pat. No. 5,474,771. The hu5c8 mAb hyridomais available from the ATCC(HB 10916). The wild-type amino acid sequencefor the light chain and heavy chain variable regions of the 5c8 antibodyare set forth in SEQ ID NO:1 and SEQ ID NO:4, respectively.

In one aspect, the present invention provides 5c8 antibody derivativescomprising at least one amino acid substitution in a variable region ofthe light chain. The wild type 5c8 light chain variable region is setforth in SEQ ID NO:1 (amino acid sequence). The present inventionprovides peptides comprising the wild type 5c8 light chain variableregion wherein at least one amino acid substitution is made (i.e, thepeptides do not comprise SEQ ID NO:1). The light chain peptide cancomprise the entire light chain, including both the constant region andthe variable region, or may comprise only the variable region. The lightchain peptide can also comprise the entire variable region and a portionof the constant region, which can range from the entire constant regionless one amino acid to just one amino acid of the constant region, orany range therewithin. Alternately, the light chain peptide can comprisethe variable region fused to any unrelated peptide sequence, thusforming a fusion protein.

In some embodiments, the light chain peptide comprises at least oneamino acid substitution in any of the following positions of the wildtype 5c8 antibody light chain variable region (see SEQ ID NO:1):positions selected from the group consisting of 24, 26, 27, 28, 30, 31,32, 33, 34, 35, 36, 38, 54, 57, 58, 59, 60, 93, 95, 96, 97, 98, 100, and101, or any subset thereof. Such a peptide comprising these positionswhich may contain an amino acid substitution is set forth in SEQ ID NO:3(amino acid sequence). These light chain peptides can also comprise atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, or at least 23 aminoacid substitutions, or a substitution in each of the 24 positions.

In some embodiments, the light chain peptide comprises at least oneamino acid substitution in any of positions 26, 27, 31, 32, 33, and 98,or any subset thereof. Such a peptide comprising these positions whichmay contain an amino acid substitution is set forth in SEQ ID NO:3(amino acid sequence), wherein positions 24, 28, 30, 34, 35, 36, 38, 54,57, 58, 59, 60, 93, 95, 96, 97, 100, and 101 are wild type amino acids.These peptides can also comprise at least 2, at least 3, at least 4, orat least 5 amino acid substitutions, or a substitution in each of the 6positions.

The substituted amino acid in each of the foregoing positions (i.e.,positions 24, 26, 27, 28, 30, 31, 32, 33, 34, 35, 36, 38, 54, 57, 58,59, 60, 93, 95, 96, 97, 98, 100, and 101) can each be, independently,any naturally occurring amino acid or any non-naturally occurring aminoacid. Thus, a particular light chain peptide may comprise one or moreamino acid substitutions that are naturally occurring amino acids and/orone or more amino acid substitutions that are non-naturally occurringamino acids.

Individual amino acid substitutions are selected from any one of thefollowing: 1) the set of amino acids with nonpolar sidechains, forexample, Ala, Cys, Ile, Leu, Met, Phe, Pro, Val; 2) the set of aminoacids with negatively charged side chains, for example, Asp, Glu; 3) theset of amino acids with positively charged sidechains, for example, Arg,His, Lys; and 4) the set of amino acids with uncharged polar sidechains,for example, Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, Tyr, towhich are added Cys, Gly, Met and Phe.

Naturally occurring amino acids include, for example, alanine (Ala),arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys),glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His),isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met),phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),tryptophan (Trp), tyrosine (Tyr), and valine (Val).

Non-naturally occurring amino acids include, for example, norleucine,ornithine, norvaline, homoserine, and other amino acid residue analoguessuch as those described in Ellman et al., Meth. Enzym., 1991, 202,301-336. To generate such non-naturally occurring amino acid residues,the procedures of Noren et al., Science, 1989, 244, 182 and Ellman etal., supra, can be used. Briefly, these procedures involve chemicallyactivating a suppressor tRNA with a non-naturally occurring amino acidresidue followed by in vitro transcription and translation of the RNA.

In some embodiments, the light chain peptide comprises an amino acidsequence that is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, or at least 95% identical to a corresponding region of SEQID NO:3. Thus, for example, a light chain peptide that comprises twoamino acid substitutions at positions 26 and 27 can still haveadditional substitutions elsewhere in the peptide (i.e., at positionsother than those denoted “Xaa” in SEQ ID NO:3) so long as the peptide isat least within the stated percentage of being identical. Thus, peptideshaving additional amino acid substitutions or amino acid insertions ordeletions are contemplated herein. It is to be understood that whendetermining the percent identity of a peptide that comprises a portionof the constant region of the light chain, only the variable region isconsidered for percent identity purposes (i.e, the constant regionportion is not included within the percent identity).

The fragment can comprise at least 8, at least 10, at least 15, at least20, at least 25, at least 30, at least 35, at least 40, at least 45, atleast 50, at least 55, at least 60, at least 65, at least 70, at least75, at least 80, at least 85, at least 90, at least 95, at least 100, orat least 105 amino acids, with a maximum of 110 amino acids. In someembodiments, the fragment comprises at least one of positions 24, 26,27, 28, 30, 31, 32, 33, 34, 35, 36, 38, 54, 57, 58, 59, 60, 93, 95, 96,97, 98, 100, and 101, or any subset thereof, referring to SEQ ID NO:3.In some embodiments, the fragment comprises one or more of any of theforegoing light chain amino acid substitutions.

The light chain peptides, and fragments thereof, described herein retaintheir ability to bind to CD154. That is, replacement of thecorresponding wild type amino acids of 5c8 antibody with the peptides orfragments described herein will still allow the resultant antibody tobind human CD 154. For example, replacement of all or a portion of thewild type 5c8 light chain variable region amino acids with thecorresponding amino acids of a peptide or fragment described herein willresult in an antibody that retains the ability to bind human CD154.

In some embodiments, the light chain may be substituted with thefollowing types of amino acids at the indicated positions: R24(negative, positive, polar, nonpolar); S26 (negative, polar, nonpolar);Q27 (positive, negative, polar); R28 (negative, polar, nonpolar); S30(positive, negative, polar); S31 (positive, polar, nonpolar); S32(positive, polar, nonpolar); T33 (positive, negative, polar, nonpolar);Y34 (positive, negative, polar, nonpolar); S35 (polar); Y 36 (polar,nonpolar, negative); H38 (positive, negative, polar, nonpolar); Y54(negative, polar, nonpolar); N57 (negative, polar); L58 (negative,polar); E59 (negative, polar); S60 (negative); Q93 (positive, negative,polar, nonpolar); S95 (negative, polar); W96 (negative, positive,polar); E97 (negative); 198 (negative, polar, nonpolar); P100 (negative,polar); and T101 (negative, polar, nonpolar).

In some embodiments, the amino acid at position 24 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Arg, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Trp, Tyr, Ala, Ile, Leu, Pro, Val, Asp, Glu, and Lys, or any subsetthereof. The amino acid at position 24 can also be preferably Arg.

In some embodiments, the amino acid at position 26 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Ser, Asn, Cys, Gln, Gly, His, Met, Phe, Thr,Trp, Tyr, Ala, Ile, Leu, Pro, Val, Asp, and Glu, or any subset thereof.Alternately, the amino acid at position 26 are preferably Ser or Asp.The amino acid at position 26 can also be preferably Ser, Asp or Glu; orpreferably Ser or Asp.

In some embodiments, the amino acid at position 27 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Gln, Asn, Cys, Gly, His, Met, Phe, Ser, Thr,Trp, Tyr, Asp, Glu, Arg, and Lys, or any subset thereof. Alternately,the amino acid at position 27 are preferably Gln or Glu. The amino acidat position 27 can also be preferably Gln, Asp, or Glu; or preferablyGln or Glu.

In some embodiments, the amino acid at position 28 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Arg, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Trp, Tyr, Asp, Glu, Ala, Ile, Leu, Pro, and Val, or any subsetthereof. Alternately, the amino acid at position 28 are preferably Argor Glu. The amino acid at position 28 can also be preferably Arg.

In some embodiments, the amino acid at position 30 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Ser, Asn, Cys, Gln, Gly, His, Met, Phe, Thr,Trp, Tyr, Asp, Glu, Arg, and Lys, or any subset thereof. The amino acidat position 30 can also be preferably Ser.

In some embodiments, the amino acid at position 31 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Ser, Asn, Cys, Gln, Gly, His, Met, Phe, Thr,Trp, Tyr, Ala, Ile, Leu, Pro, Val, Arg, and Lys, or any subset thereof.Alternately, the amino acid at position 31 is selected from the groupconsisting of Ser, Ala, His, Asn, Thr, Val, and Trp. The amino acid atposition 31 is also selected from the group consisting of Ser, Asn, Cys,Gln, Gly, Thr, and Tyr; or is preferably Ser or Asn.

In some embodiments, the amino acid at position 32 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Ser, Asn, Cys, Gln, Gly, His, Met, Phe, Thr,Trp, Tyr, Arg, Ala, Ile, Leu, and Lys, or any subset thereof.Alternately, the amino acid at position 32 is selected from the groupconsisting of Ser, Ala, Phe, Ile, Leu, Met, and Trp. The amino acid atposition 32 is also selected from the group consisting of Ser, Ala, Ile,Leu, Met, Phe, Pro, Val, and Trp; or is preferably Ser or Phe.

In some embodiments, the amino acid at position 33 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Thr, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Trp, Tyr, Asp, Glu, Arg, Ala, Val, and Lys, or any subset thereof.Alternately, the amino acid at position 33 is selected from the groupconsisting of Thr, Ala, Phe, Met, Val, Trp, Asp, Arg, Tyr, and Gln. Theamino acid at position 33 is also selected from the group consisting ofThr, Asn, Cys, Gln, Gly, Ser, and Tyr; or is preferably Thr, Gln, orTyr.

In some embodiments, the amino acid at position 34 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Tyr, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Trp Ala, Ile, Leu, Pro, Val, Asp, Glu, Arg, and Lys, or any subsetthereof. Alternately, the amino acid at position 34 is selected from thegroup consisting of Tyr, Ala, Asp, Glu, Phe, Ile, Lys, Leu, Met, Arg,Val, and Trp. The amino acid at position 34 can also be preferably Tyr.

In some embodiments, the amino acid at position 35 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Ser, Asn, Cys, Gln, Gly, His, Met, Phe, Thr,Trp, and Tyr, or any subset thereof. The amino acid at position 35 canalso be preferably Ser.

In some embodiments, the amino acid at position 36 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Tyr, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Trp Asp, Ala, Leu, and Glu, or any subset thereof. Alternately, theamino acid at position 36 is selected from the group consisting of Tyr,Ala, Phe, Leu, and Trp. The amino acid at position 36 can also bepreferably Tyr.

In some embodiments, the amino acid at position 38 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of His, Asn, Cys, Gln, Gly, Met, Phe, Ser, Thr,Trp, Tyr, Ala, Ile, Leu, Pro, Val, Asp, Glu, Arg, and Lys, or any subsetthereof. The amino acid at position 38 can also be preferably His.

In some embodiments, the amino acid at position 54 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Tyr, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Trp Ala, Ile, Leu, Pro, Val, Asp, and Glu, or any subset thereof.Alternately, the amino acid at position 54 are preferably Tyr or Glu.The amino acid at position 54 can also be preferably Tyr.

In some embodiments, the amino acid at position 57 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr,Trp, Tyr, Asp, and Glu, or any subset thereof. The amino acid atposition 57 can also be preferably Asn.

In some embodiments, the amino acid at position 58 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Leu, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Trp Asp, and Glu, or any subset thereof. The amino acid at position58 can also be preferably Leu.

In some embodiments, the amino acid at position 59 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Glu, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Trp, Tyr, and Asp, or any subset thereof. The amino acid atposition 59 can also be preferably Glu.

In some embodiments, the amino acid at position 60 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Ser, Asp, and Glu, or any subset thereof. Theamino acid at position 60 can also be preferably Ser.

In some embodiments, the amino acid at position 93 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Gln, Asn, Cys, Gly, His, Met, Phe, Ser, Thr,Trp, Tyr, Ala, Ile, Leu, Pro, Val, Asp, Glu, Arg, and Lys, or any subsetthereof. The amino acid at position 93 can also be preferably Gln.

In some embodiments, the amino acid at position 95 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Ser, Asn, Cys, Gln, Gly, His, Met, Phe, Thr,Trp, Tyr, Asp, and Glu, or any subset thereof. The amino acid atposition 95 can also be preferably Ser.

In some embodiments, the amino acid at position 96 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Trp, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Tyr, Asp, Glu, Arg, and Lys, or any subset thereof. Alternately,the amino acid at position 96 is selected from the group consisting ofTrp, Asp, Glu, His, Arg, Ser, and Thr. The amino acid at position 96 canalso be preferably Trp.

In some embodiments, the amino acid at position 97 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is Glu or Asp, orany subset thereof. The amino acid at position 97 can also be preferablyGlu.

In some embodiments, the amino acid at position 98 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Ile, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Trp, Tyr, Asp, Ala, Leu, Pro, Val, and Glu, or any subset thereof.Alternately, the amino acid at position 98 selected from the groupconsisting of Ile, Ala, Phe, His, Leu, Met, Asn, Pro, Gln, Ser, Thr,Val, Trp, and Tyr. The amino acid at position 98 is selected from thegroup consisting of Ile, Asn, Cys, Gln, Gly, Ser, Thr, and Tyr; orpreferably Ile or Gln.

In some embodiments, the amino acid at position 100 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Pro, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Trp, Tyr, Asp, and Glu, or any subset thereof. The amino acid atposition 100 can also be preferably Pro.

In some embodiments, the amino acid at position 101 (referring to SEQ IDNO:3) of a light chain peptide, or fragment thereof, is selected fromthe group consisting of Thr, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Trp, Tyr, Ala, Ile, Leu, Pro, Val, Asp, and Glu, or any subset thereof.The amino acid at position 101 can also be preferably Thr.

In some embodiments, the amino acid at position is 26 Asp; the aminoacid at position 27 is Glu; the amino acid at position 28 is Glu; theamino acid at position 31 is Val; the amino acid at position 33 is Aspor Arg; and the amino acid at position 54 is Glu.

In some embodiments, the amino acid at position 31 is His or Asn; theamino acid at position 32 is Trp or Phe; the amino acid at position 33is Trp, Tyr, or Gln; the amino acid at position 36 is Leu or Trp; theamino acid at position 96 is His; and the amino acid at position 98 isPhe or Gln.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Gln; theamino acid at position 31 and position 32 is Ser; the amino acid atposition 33 is Thr; the amino acid at position 98 is Ile; the amino acidat position 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr. This mutant is referred to as Mutant #6 herein.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26, position 31, and position 32 is Ser; the amino acidat position 27 is Glu; the amino acid at position 33 is Thr; the aminoacid at position 98 is Ile; the amino acid at position 24 and position28 is Arg; the amino acid at position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 34, position 36, andposition 54 is Tyr; the amino acid at position 38 is His; the amino acidat position 57 is Asn; the amino acid at position 58 is Leu; the aminoacid at position 59 and position 97 is Glu; the amino acid at position93 is Gln; the amino acid at position 96 is Trp; the amino acid atposition 100 is Pro; and the amino acid at position 101 is Thr. Thismutant is referred to as mutant #7 herein.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Glu; theamino acid at position 31 and position 32 is Ser; the amino acid atposition 33 is Thr; the amino acid at position 98 is Ile; the amino acidat position 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr. This mutant is referred to as Mutant #14 herein.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 27 and position 93 is Gln; the amino acid at position26, position 31, position 32, position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 33 and position 101 isThr; the amino acid at position 98 is Ile; the amino acid at position 24and position 28 is Arg; the amino acid at position 34 and position 54 isTyr; the amino acid at position 38 is His; the amino acid at position 57is Asn; the amino acid at position 58 is Leu; the amino acid at position59 and position 97 is Glu; the amino acid at position 96 and position 36is Trp; and the amino acid at position 100 is Pro. This mutant isreferred to as Mutant #3 herein.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 27 and position 93 is Gln; the amino acid at position26, position 31, position 32, position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 101 is Thr; the aminoacid at position 98 is Ile; the amino acid at position 24 and position28 is Arg; the amino acid at position 34, position 36, and position 54is Tyr; the amino acid at position 38 is His; the amino acid at position57 is Asn; the amino acid at position 58 is Leu; the amino acid atposition 59 and position 97 is Glu; the amino acid at position 96 andposition 33 is Trp; and the amino acid at position 100 is Pro. Thismutant is referred to as Mutant #5 herein.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 27 and position 93 is Gln; the amino acid at position26, position 32, position 30, position 35, position 60, and position 95is Ser; the amino acid at position 33 and position 101 is Thr; the aminoacid at position 98 is Ile; the amino acid at position 24 and position28 is Arg; the amino acid at position 34, position 36, and position 54is Tyr; the amino acid at position 38 is His; the amino acid at position57 is Asn; the amino acid at position 58 is Leu; the amino acid atposition 59 and position 97 is Glu; the amino acid at position 96 isTrp; the amino acid at position 31 is Val; and the amino acid atposition 100 is Pro. This mutant is referred to as Mutant #8 herein.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 27 and position 93 is Gln; the amino acid at position26, position 31, position 32, position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 101 is Thr; the aminoacid at position 98 is Ile; the amino acid at position 24 and position28 is Arg; the amino acid at position 34, position 36, and position 54is Tyr; the amino acid at position 38 is His; the amino acid at position57 is Asn; the amino acid at position 58 is Leu; the amino acid atposition 59 and position 97 is Glu; the amino acid at position 96 isTrp; the amino acid at position 33 is Asp; and the amino acid atposition 100 is Pro. This mutant is referred to as Mutant #9 herein.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 27 and position 93 is Gln; the amino acid at position26, position 31, position 32, position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 33 and position 101 isThr; the amino acid at position 98 is Ile; the amino acid at position 24and position 28 is Arg; the amino acid at position 34 and position 36 isTyr; the amino acid at position 38 is His; the amino acid at position 57is Asn; the amino acid at position 58 is Leu; the amino acid at position54, position 59 and position 97 is Glu; the amino acid at position 96 isTrp; and the amino acid at position 100 is Pro. This mutant is referredto as Mutant #10 herein.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Glu; theamino acid at position 31 and position 32 is Ser; the amino acid atposition 98 is Ile; the amino acid at position 24 and position 28 isArg; the amino acid at position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 34, position 36, andposition 54 is Tyr; the amino acid at position 38 is His; the amino acidat position 57 is Asn; the amino acid at position 58 is Leu; the aminoacid at position 59 and position 97 is Glu; the amino acid at position93 is Gln; the amino acid at position 33 and position 96 is Trp; theamino acid at position 100 is Pro; and the amino acid at position 101 isThr. This mutant is referred to as Mutant #15 herein.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Gln; theamino acid at position 31 and position 32 is Ser; the amino acid atposition 33 is Thr; the amino acid at position 98 is Ile; the amino acidat position 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26, position 31, and position 32 is Ser; the amino acidat position 27 is Glu; the amino acid at position 33 is Thr; the aminoacid at position 98 is Ile; the amino acid at position 24 and position28 is Arg; the amino acid at position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 34, position 36, andposition 54 is Tyr; the amino acid at position 38 is His; the amino acidat position 57 is Asn; the amino acid at position 58 is Leu; the aminoacid at position 59 and position 97 is Glu; the amino acid at position93 is Gln; the amino acid at position 96 is Trp; the amino acid atposition 100 is Pro; and the amino acid at position 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 and position 32 is Ser; the amino acid at position27 is Gln; the amino acid at position 31 is Asn; the amino acid atposition 33 is Thr; the amino acid at position 98 is Ile; the amino acidat position 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 and position 31 is Ser; the amino acid at position27 is Gln; the amino acid at position 32 is Phe; the amino acid atposition 33 is Thr; the amino acid at position 98 is Ile; the amino acidat position 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26, position 31, and position 32 is Ser; the amino acidat position 27 and position 33 is Gln; the amino acid at position 98 isIle; the amino acid at position 24 and position 28 is Arg; the aminoacid at position 30, position 35, position 60, and position 95 is Ser;the amino acid at position 34, position 36, and position 54 is Tyr; theamino acid at position 38 is His; the amino acid at position 57 is Asn;the amino acid at position 58 is Leu; the amino acid at position 59 andposition 97 is Glu; the amino acid at position 93 is Gln; the amino acidat position 96 is Trp; the amino acid at position 100 is Pro; and theamino acid at position 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26, position 31, and position 32 is Ser; the amino acidat position 27 is Gln; the amino acid at position 33 is Tyr; the aminoacid at position 98 is Ile; the amino acid at position 24 and position28 is Arg; the amino acid at position 30, position 35, position 60, andposition 95 is Ser; the amino acid at position 34, position 36, andposition 54 is Tyr; the amino acid at position 38 is His; the amino acidat position 57 is Asn; the amino acid at position 58 is Leu; the aminoacid at position 59 and position 97 is Glu; the amino acid at position93 is Gln; the amino acid at position 96 is Trp; the amino acid atposition 100 is Pro; and the amino acid at position 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26, position 31, and position 32 is Ser; the amino acidat position 27 and position 98 is Gln; the amino acid at position 33 isThr; the amino acid at position 24 and position 28 is Arg; the aminoacid at position 30, position 35, position 60, and position 95 is Ser;the amino acid at position 34, position 36, and position 54 is Tyr; theamino acid at position 38 is His; the amino acid at position 57 is Asn;the amino acid at position 58 is Leu; the amino acid at position 59 andposition 97 is Glu; the amino acid at position 93 is Gln; the amino acidat position 96 is Trp; the amino acid at position 100 is Pro; and theamino acid at position 101 is Thr.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Glu; theamino acid at position 31 is Asn; the amino acid at position 32 is Ser;the amino acid at position 33 is Thr; the amino acid at position 98 isIle; the amino acid at position 24 and position 28 is Arg; the aminoacid at position 30, position 35, position 60, and position 95 is Ser;the amino acid at position 34, position 36, and position 54 is Tyr; theamino acid at position 38 is His; the amino acid at position 57 is Asn;the amino acid at position 58 is Leu; the amino acid at position 59 andposition 97 is Glu; the amino acid at position 93 is Gln; the amino acidat position 96 is Trp; the amino acid at position 100 is Pro; and theamino acid at position 101 is Thr. This peptide is also referred toherein as the “S26D/Q27E/S31N” peptide.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Glu; theamino acid at position 31 is Ser; the amino acid at position 32 is Phe;the amino acid at position 33 is Thr; the amino acid at position 98 isIle; the amino acid at position 24 and position 28 is Arg; the aminoacid at position 30, position 35, position 60, and position 95 is Ser;the amino acid at position 34, position 36, and position 54 is Tyr; theamino acid at position 38 is His; the amino acid at position 57 is Asn;the amino acid at position 58 is Leu; the amino acid at position 59 andposition 97 is Glu; the amino acid at position 93 is Gln; the amino acidat position 96 is Trp; the amino acid at position 100 is Pro; and theamino acid at position 101 is Thr. This peptide is also referred toherein as the “S26D/Q27E/S32F” peptide.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Glu; theamino acid at position 31 and position 32 is Ser; the amino acid atposition 33 is Gln; the amino acid at position 98 is Ile; the amino acidat position 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr. This peptide is also referred to herein as the“S26D/Q27E/T33Q” peptide.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Glu; theamino acid at position 31 and position 32 is Ser; the amino acid atposition 33 is Tyr; the amino acid at position 98 is Ile; the amino acidat position 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr. This peptide is also referred to herein as the“S26D/Q27E/T33Y” peptide.

In some embodiments, the peptide comprises SEQ ID NO:3 wherein the aminoacid at position 26 is Asp; the amino acid at position 27 is Glu; theamino acid at position 31 and position 32 is Ser; the amino acid atposition 33 is Thr; the amino acid at position 98 is Gln; the amino acidat position 24 and position 28 is Arg; the amino acid at position 30,position 35, position 60, and position 95 is Ser; the amino acid atposition 34, position 36, and position 54 is Tyr; the amino acid atposition 38 is His; the amino acid at position 57 is Asn; the amino acidat position 58 is Leu; the amino acid at position 59 and position 97 isGlu; the amino acid at position 93 is Gln; the amino acid at position 96is Trp; the amino acid at position 100 is Pro; and the amino acid atposition 101 is Thr. This peptide is also referred to herein as the“S26D/Q27E/198Q” peptide.

In another aspect, the present invention provides 5c8 antibodyderivatives comprising at least one amino acid substitution in avariable region of the heavy chain. The wild type 5c8 heavy chainvariable region is set forth in SEQ ID NO:4 (amino acid sequence). Thepresent invention provides peptides comprising the wild type 5c8 heavychain variable region wherein at least one amino acid substitution ismade (i.e, the peptides do not comprise SEQ ID NO:4). The heavy chainpeptide can comprise the entire heavy chain, including both the constantregion (CH1 and/or CH2 and/or CH3) and the variable region, or maycomprise only the variable region. The heavy chain peptide can alsocomprise the entire variable region and a portion of the constantregion, which can range from the entire constant region less one aminoacid to just one amino acid of the constant region, or any rangetherewithin. Alternately, the heavy chain peptide can comprise thevariable region fused to any unrelated peptide sequence, thus forming afusion protein.

In some embodiments, the heavy chain peptide comprises at least oneamino acid substitution in any of the following positions of the wildtype 5c8 antibody heavy chain variable region (see SEQ ID NO:4):positions selected from the group consisting of 28, 30, 31, 32, 33, 35,50, 52, 53, 54, 55, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 99, 100,101, 102, 103, 104, 105, 106, and 107, or any subset thereof. Such apeptide comprising these positions which may contain an amino acidsubstitution is set forth in SEQ ID NO:6 (amino acid sequence). Theseheavy chain peptides can also comprise at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, or at least 29 amino acid substitutions, or anamino acid substitution in each of the 30 positions.

In some embodiments, the heavy chain peptide comprises at least oneamino acid substitution in any of positions 31, 54, 57, 101, and 103, orany subset thereof. Such a peptide comprising these positions which maycontain an amino acid substitution is set forth in SEQ ID NO:6 (aminoacid sequence), wherein positions 28, 30, 32, 33, 35, 50, 52, 53, 55,58, 59, 60, 61, 62, 63, 64, 65, 66, 99, 100, 102, 104, 105, 106, and 107are wild type amino acids. These peptides can also comprise at least 2,at least 3, at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 21, at least 22, at least 23, or at least 24 aminoacid substitutions, or an amino acid substitution in each of the 25positions.

The substituted amino acid in each of the foregoing positions (i.e.,positions 28, 30, 31, 32, 33, 35, 50, 52, 53, 54, 55, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 99, 100, 101, 102, 103, 104, 105, 106, and 107)can each be, independently, any naturally occurring amino acid or anynon-naturally occurring amino acid. Thus, a particular heavy chainpeptide may comprise one or more amino acid substitutions that arenaturally occurring amino acids and/or one or more amino acidsubstitutions that are non-naturally occurring amino acids.

In some embodiments, the heavy chain peptide comprises an amino acidsequence that is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, or at least 95% identical to SEQ ID NO:6. Thus, forexample, a heavy chain peptide that comprises an amino acid substitutionat position 31 can still have additional substitutions elsewhere in thepeptide (i.e., at positions other than those denoted “Xaa” in SEQ IDNO:6) so long as the peptide is at least within the stated percentage ofbeing identical. Thus, peptides having additional amino acidsubstitutions or amino acid insertions or deletions are contemplatedherein. It is to be understood that when determining the percentidentity of a peptide that comprises a portion of the constant region ofthe heavy chain, only the variable region is considered for percentidentity purposes (i.e, the constant region portion is not includedwithin the percent identity).

In some embodiments, a fragment of a heavy chain peptide that comprisesan amino acid sequence at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% identical to a correspondingregion of SEQ ID NO:6 is provided.

The fragment can comprise at least 8, at least 10, at least 15, at least20, at least 25, at least 30, at least 35, at least 40, at least 45, atleast 50, at least 55, at least 60, at least 65, at least 70, at least75, at least 80, at least 85, at least 90, at least 95, at least 100, atleast 105, at least 110, or at least 115 amino acids, with a maximum of117 amino acids. In some embodiments, the fragment comprises at leastone of positions 28, 30, 31, 32, 33, 35, 50, 52, 53, 54, 55, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 99, 100, 101, 102, 103, 104, 105, 106, and107, or any subset thereof, referring to SEQ ID NO:6. In someembodiments, the fragment comprises one or more of any of the foregoingheavy chain amino acid substitutions.

The heavy chain peptides, and fragments thereof, described herein retaintheir ability to bind to CD154. That is, replacement of thecorresponding wild type amino acids of 5c8 antibody with the peptides orfragments described herein will still allow the resultant antibody tobind human CD 154. For example, replacement of all or a portion of thewild type 5c8 heavy chain variable region amino acids with thecorresponding amino acids of a peptide or fragment described herein willresult in an antibody that retains the ability to bind human CD154. Insome embodiments, the antibodies described herein may contact one ormore less residues of CD154 than the wild type 5c8 antibody.

In some embodiments, the heavy chain may be substituted with thefollowing types of amino acids at the indicated positions: I28(positive, negative, polar); T30 (positive, negative, polar, nonpolar);S31 (positive, polar); Y32 (positive, negative, polar, nonpolar); Y33(positive, negative, polar, nonpolar); Y35 (polar); E50 (negative); N52(polar, nonpolar); P53 (negative, polar); S54 (positive, negative,polar); N55 (negative, polar, nonpolar); D57 (negative, nonpolar); T58(polar, nonpolar); N59 (negative, polar, nonpolar); F60 (negative,positive, polar, nonpolar); N61 (negative); E62 (negative); K63(negative, positive, polar, nonpolar); F64 (negative, polar, nonpolar);K65 (negative, positive, polar, nonpolar); S66 (negative); S99(negative, nonpolar); D100 (negative); G101 (nonpolar); R102 (positive,polar); N103 (negative, positive, polar, nonpolar); D104 (negative);M105 (negative, polar, positive); D106 (negative); and S107 (negative,polar, nonpolar).

In some embodiments, the amino acid at position 28 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Ile, Asp, Glu, Arg, Lys, Asn, Cys, Gln, Gly,His, Met, Phe, Ser, Thr, and Trp, or any subset thereof. Alternately,the amino acid at position 28 is selected from the group consisting ofIle, His, Asn, Gln, Ser, Thr, and Glu. The amino acid at position 28 canalso be preferably Ile.

In some embodiments, the amino acid at position 30 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Thr, Asp, Glu, Arg, Lys, Asn, Cys, Gln, Gly,His, Met, Phe, Ser, Trp, Tyr, Ala, Ile, Leu, Pro, and Val, or any subsetthereof. Alternately, the amino acid at position 30 is selected from thegroup consisting of Thr, His, Asn, Gln, Ser, Tyr, and Arg. The aminoacid at position 30 can also be preferably Thr.

In some embodiments, the amino acid at position 31 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Ser, Arg, His, Lys, Gln, and Trp, or any subsetthereof. Alternately, the amino acid at position 31 are preferably Ser,Gln, or Trp. The amino acid at position 31 selected from the groupconsisting of Ser, Ala, Ile, Leu, Met, Phe, Pro, Val, Trp, Asn, Cys,Gln, Gly, Ser, Thr, and Tyr; or preferably is Ser, Trp, or Gln.

In some embodiments, the amino acid at position 32 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Tyr, Asp, Glu, Arg, Lys, Asn, Cys, Gln, Gly,His, Met, Phe, Ser, Trp, Ala, Ile, Leu, Pro, and Val, or any subsetthereof. The amino acid at position 32 can also be preferably Tyr.

In some embodiments, the amino acid at position 33 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Tyr, Asp, Glu, Asn, Cys, Gln, Gly, His, Met,Phe, Ser, Thr, Trp Ala, Ile, Leu, Pro, and Val, or any subset thereof.Alternately, the amino acid at position 33 is selected from the groupconsisting of Tyr, Ala, Pro, Ser, Thr, Val, and Trp. The amino acid atposition 33 can also be preferably Tyr.

In some embodiments, the amino acid at position 35 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Tyr, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Trp Asp, and Glu, or any subset thereof. The amino acid at position35 can also be preferably Tyr.

In some embodiments, the amino acid at position 50 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is Glu or Asp, orany subset thereof. The amino acid at position 50 can also be preferablyGlu.

In some embodiments, the amino acid at position 52 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr,Trp, Tyr, Arg, Ala, Leu, Val, and Lys, or any subset thereof.Alternately, the amino acid at position 52 is selected from the groupconsisting of Asn, Ala, Phe, His, Leu, Met, Ser, Thr, Val, and Trp. Theamino acid at position 52 can also be preferably Asn.

In some embodiments, the amino acid at position 53 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Pro, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Trp, Tyr, Asp, and Glu, or any subset thereof. The amino acid atposition 53 can also be preferably Pro.

In some embodiments, the amino acid at position 54 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Ser, Asn, Cys, Gln, Gly, His, Met, Phe, Thr,Trp, Tyr, Arg, Lys, and Glu, or any subset thereof. Alternately, theamino acid at position 54 is selected from the group consisting of Ser,Glu, His, Lys, Asn, Gln, Arg, Thr, Trp, Tyr, and Phe. The amino acid atposition 54 is also selected from the group consisting of Ser, Ala, Ile,Leu, Met, Phe, Pro, Val, Trp, Asn, Cys, Gln, Gly, Ser, Thr, and Tyr; orpreferably Ser, Phe, or Gln.

In some embodiments, the amino acid at position 55 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Asn, Glu, Lys, Gln, Ser, Thr, Met, and Val, orany subset thereof. Alternately, the amino acid at position 55 selectedfrom the group consisting of Asn, Glu, Lys, Gln, Ser, Thr, Met, and Val.The amino acid at position 55 can also be preferably Asn.

In some embodiments, the amino acid at position 57 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Asp, Glu, Phe, and Leu, or any subset thereof.Alternately, the amino acid at position 57 are preferably Asp, Phe, orLeu. The amino acid at position 57 selected from the group consisting ofAsp, Ala, Ile, Leu, Met, Phe, Pro, Val, and Trp; or is preferably Asp,Leu, or Phe.

In some embodiments, the amino acid at position 58 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Thr, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Trp, Tyr, Ala, Ile, Leu, Pro, and Val, or any subset thereof. The aminoacid at position 58 can also be preferably Thr.

In some embodiments, the amino acid at position 59 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr,Trp, Tyr, Ala, Ile, Leu, Pro, Val, Asp, and Glu, or any subset thereof.Alternately, the amino acid at position 59 selected from the groupconsisting of Asn, Ala, Phe, Leu, Met, Pro, Val, Trp, Asp, and Tyr. Theamino acid at position 59 can also be preferably Asn.

In some embodiments, the amino acid at position 60 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Phe, Asp, Asn, Cys, Gln, Gly, His, Met, Ser,Thr, Trp, Tyr, Asp, Glu, Arg, Lys, Ala, Ile, Leu, Pro, and Val, or anysubset thereof. The amino acid at position 60 can also be preferablyPhe.

In some embodiments, the amino acid at position 61 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is Asn, Asp, orGlu, or any subset thereof. The amino acid at position 61 can also bepreferably Asn.

In some embodiments, the amino acid at position 62 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is Glu or Asp, orany subset thereof. The amino acid at position 62 can also be preferablyGlu.

In some embodiments, the amino acid at position 63 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Lys, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Trp, Tyr, Arg, Glu, Asp, Ala, Ile, Leu, Pro, and Val, or any subsetthereof. The amino acid at position 63 can also be preferably Lys.

In some embodiments, the amino acid at position 64 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Phe, Asn, Cys, Gln, Gly, His, Met, Ser, Thr,Trp, Tyr, Asp, Glu, Ala, Ile, Leu, Pro, and Val, or any subset thereof.The amino acid at position 64 can also be preferably Phe.

In some embodiments, the amino acid at position 65 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Lys, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Trp, Tyr, Arg, Asp, Glu, Ala, Ile, Leu, Pro, and Val, or any subsetthereof. The amino acid at position 65 can also be preferably Lys.

In some embodiments, the amino acid at position 66 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is Ser, Asp, orGlu, or any subset thereof. The amino acid at position 66 can also bepreferably Ser.

In some embodiments, the amino acid at position 99 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is Ser, Asp, Glu,or Ala, or any subset thereof. Alternately, the amino acid at position99 are preferably Ser or Ala. The amino acid at position 99 can also bepreferably Ser.

In some embodiments, the amino acid at position 100 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is Asp or Glu, orany subset thereof. The amino acid at position 100 can also bepreferably Asp.

In some embodiments, the amino acid at position 101 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is Gly, Phe, orLeu, or any subset thereof. Alternately, the amino acid at position 101are preferably Gly, Phe, or Leu. The amino acid at position 101 selectedfrom the group consisting of Gly, Ala, Ile, Leu, Met, Phe, Pro, Val, andTrp; or is preferably Gly, Leu, or Phe.

In some embodiments, the amino acid at position 102 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Arg, Asn, Cys, Gln, Gly, His, Met, Phe, Ser,Thr, Trp, Tyr, and Lys, or any subset thereof. The amino acid atposition 102 can also be preferably Arg.

In some embodiments, the amino acid at position 103 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr,Trp, Tyr, Asp, Glu, Ala, Ile, Lys, Arg, and Val, or any subset thereof.Alternately, the amino acid at position 103 is selected from the groupconsisting of Asn, Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Arg, Ser,Thr, Val, and Tyr. The amino acid at position 103 is also selected fromthe group consisting of Asn, Cys, Gln, Gly, Ser, Thr, and Tyr; orpreferably Asn or Tyr.

In some embodiments, the amino acid at position 104 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is Asp or Glu, orany subset thereof. The amino acid at position 104 can also bepreferably Asp.

In some embodiments, the amino acid at position 105 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Met, Asp, Glu, Arg, His, Lys, Asn, Cys, Gln,Gly, Phe, Ser, Thr, Trp, and Tyr, or any subset thereof. The amino acidat position 105 can also be preferably Met.

In some embodiments, the amino acid at position 106 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is Asp or Glu, orany subset thereof. The amino acid at position 106 can also bepreferably Asp.

In some embodiments, the amino acid at position 107 (referring to SEQ IDNO:6) of a heavy chain peptide, or fragment thereof, is selected fromthe group consisting of Ser, Asn, Cys, Gln, Gly, His, Met, Phe, Thr,Trp, Tyr, Asp, Glu, Ala, Ile, Leu, Pro, and Val, or any subset thereof.The amino acid at position 107 can also be preferably Ser.

In some embodiments, the amino acid at position 28 is Glu; the aminoacid at position 33 is Phe; the amino acid at position 54 is Thr; andthe amino acid at position 59 is Asp or Leu.

In some embodiments, the amino acid at position 30 is His or Arg; theamino acid at position 31 is Gln or Trp; the amino acid at position 33is Trp, Val, or Pro; the amino acid at position 52 is Met or Trp; theamino acid at position 54 is Asn, Phe, or Gln; the amino acid atposition 55 is Met, Lys, or Val; the amino acid at position 57 is Phe orLeu; the amino acid at position 59 is Phe or Tyr; the amino acid atposition 101 is Phe or Leu; and the amino acid at position 103 is His orTyr.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31, position 54, position 66, position 99, and position107 is Ser; the amino acid at position 57, position 100, position 104,and position 106 is Asp; the amino acid at position 101 is Gly; theamino acid at position 103, position 52, position 55, position 59, andposition 61 is Asn; the amino acid at position 28 is Ile; the amino acidat position 58 is Thr; the amino acid at position 32, position 33, andposition 35 is Tyr; the amino acid at position 50 and position 62 isGlu; the amino acid at position 53 is Pro; the amino acid at position 60and position 64 is Phe; the amino acid at position 63 and position 65 isLys; the amino acid at position 102 is Arg; the amino acid at position30 is His; and the amino acid at position 105 is Met. This mutant isreferred to as Mutant #1 herein.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31, position 54, position 66, position 99, and position107 is Ser; the amino acid at position 57, position 100, position 104,and position 106 is Asp; the amino acid at position 101 is Gly; theamino acid at position 103, position 52, position 55, position 59, andposition 61 is Asn; the amino acid at position 28 is Ile; the amino acidat position 30 and position 58 is Thr; the amino acid at position 32,and position 35 is Tyr; the amino acid at position 50 and position 62 isGlu; the amino acid at position 53 is Pro; the amino acid at position 60and position 64 is Phe; the amino acid at position 63 and position 65 isLys; the amino acid at position 102 is Arg; the amino acid at position33 is Trp; and the amino acid at position 105 is Met. This mutant isreferred to as Mutant #2 herein.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31, position 66, position 99, and position 107 is Ser;the amino acid at position 57, position 100, position 104, and position106 is Asp; the amino acid at position 101 is Gly; the amino acid atposition 103, position 52, position 54, position 55, position 59, andposition 61 is Asn; the amino acid at position 28 is Ile; the amino acidat position 30 and position 58 is Thr; the amino acid at position 32,position 33, and position 35 is Tyr; the amino acid at position 50 andposition 62 is Glu; the amino acid at position 53 is Pro; the amino acidat position 60 and position 64 is Phe; the amino acid at position 63 andposition 65 is Lys; the amino acid at position 102 is Arg; and the aminoacid at position 105 is Met. This mutant is referred to as Mutant #4herein.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31, position 54, position 66, position 99, and position107 is Ser; the amino acid at position 57, position 100, position 104,and position 106 is Asp; the amino acid at position 101 is Gly; theamino acid at position 103, position 52, position 55, position 59, andposition 61 is Asn; the amino acid at position 28 is Ile; the amino acidat position 30 and position 58 is Thr; the amino acid at position 32,and position 35 is Tyr; the amino acid at position 50 and position 62 isGlu; the amino acid at position 53 is Pro; the amino acid at position33, position 60, and position 64 is Phe; the amino acid at position 63and position 65 is Lys; the amino acid at position 102 is Arg; and theamino acid at position 105 is Met. This mutant is referred to as Mutant#11 herein.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31, position 54, position 66, position 99, and position107 is Ser; the amino acid at position 57, position 59, position 100,position 104, and position 106 is Asp; the amino acid at position 101 isGly; the amino acid at position 103, position 52, position 55, andposition 61 is Asn; the amino acid at position 28 is Ile; the amino acidat position 30 and position 58 is Thr; the amino acid at position 32,position 33, and position 35 is Tyr; the amino acid at position 50 andposition 62 is Glu; the amino acid at position 53 is Pro; the amino acidat position 60 and position 64 is Phe; the amino acid at position 63 andposition 65 is Lys; the amino acid at position 102 is Arg; and the aminoacid at position 105 is Met. This mutant is referred to as Mutant #12herein.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31, position 54, position 66, position 99, and position107 is Ser; the amino acid at position 57, position 100, position 104,and position 106 is Asp; the amino acid at position 101 is Gly; theamino acid at position 103, position 52, position 55, and position 61 isAsn; the amino acid at position 28 is Ile; the amino acid at position 30and position 58 is Thr; the amino acid at position 32, position 33, andposition 35 is Tyr; the amino acid at position 50 and position 62 isGlu; the amino acid at position 53 is Pro; the amino acid at position 60and position 64 is Phe; the amino acid at position 63 and position 65 isLys; the amino acid at position 102 is Arg; the amino acid at position59 is Leu; and the amino acid at position 105 is Met. This mutant isreferred to as Mutant #13 herein.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 is Trp; the amino acid at position 54 is Ser; theamino acid at position 57 is Asp; the amino acid at position 101 is Gly;the amino acid at position 103 is Asn; the amino acid at position 28 isIle; the amino acid at position 30 and position 58 is Thr; the aminoacid at position 32, position 33, and position 35 is Tyr; the amino acidat position 50 and position 62 is Glu; the amino acid at position 52,position 55, position 59, and position 61 is Asn; the amino acid atposition 53 is Pro; the amino acid at position 60 and position 64 isPhe; the amino acid at position 63 and position 65 is Lys; the aminoacid at position 66, position 99, and position 107 is Ser; the aminoacid at position 100, position 104, and position 106 is Asp; the aminoacid at position 102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 is Gln; the amino acid at position 54 is Ser; theamino acid at position 57 is Asp; the amino acid at position 101 is Gly;the amino acid at position 103 is Asn; the amino acid at position 28 isIle; the amino acid at position 30 and position 58 is Thr; the aminoacid at position 32, position 33, and position 35 is Tyr; the amino acidat position 50 and position 62 is Glu; the amino acid at position 52,position 55, position 59, and position 61 is Asn; the amino acid atposition 53 is Pro; the amino acid at position 60 and position 64 isPhe; the amino acid at position 63 and position 65 is Lys; the aminoacid at position 66, position 99, and position 107 is Ser; the aminoacid at position 100, position 104, and position 106 is Asp; the aminoacid at position 102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 is Ser; the amino acid at position 54 is Phe; theamino acid at position 57 is Asp; the amino acid at position 101 is Gly;the amino acid at position 103 is Asn; the amino acid at position 28 isIle; the amino acid at position 30 and position 58 is Thr; the aminoacid at position 32, position 33, and position 35 is Tyr; the amino acidat position 50 and position 62 is Glu; the amino acid at position 52,position 55, position 59, and position 61 is Asn; the amino acid atposition 53 is Pro; the amino acid at position 60 and position 64 isPhe; the amino acid at position 63 and position 65 is Lys; the aminoacid at position 66, position 99, and position 107 is Ser; the aminoacid at position 100, position 104, and position 106 is Asp; the aminoacid at position 102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 is Ser; the amino acid at position 54 is Gln; theamino acid at position 57 is Asp; the amino acid at position 101 is Gly;the amino acid at position 103 is Asn; the amino acid at position 28 isIle; the amino acid at position 30 and position 58 is Thr; the aminoacid at position 32, position 33, and position 35 is Tyr; the amino acidat position 50 and position 62 is Glu; the amino acid at position 52,position 55, position 59, and position 61 is Asn; the amino acid atposition 53 is Pro; the amino acid at position 60 and position 64 isPhe; the amino acid at position 63 and position 65 is Lys; the aminoacid at position 66, position 99, and position 107 is Ser; the aminoacid at position 100, position 104, and position 106 is Asp; the aminoacid at position 102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 and position 54 is Ser; the amino acid at position57 is Phe; the amino acid at position 101 is Gly; the amino acid atposition 103 is Asn; the amino acid at position 28 is Ile; the aminoacid at position 30 and position 58 is Thr; the amino acid at position32, position 33, and position 35 is Tyr; the amino acid at position 50and position 62 is Glu; the amino acid at position 52, position 55,position 59, and position 61 is Asn; the amino acid at position 53 isPro; the amino acid at position 60 and position 64 is Phe; the aminoacid at position 63 and position 65 is Lys; the amino acid at position66, position 99, and position 107 is Ser; the amino acid at position100, position 104, and position 106 is Asp; the amino acid at position102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 and position 54 is Ser; the amino acid at position57 is Leu; the amino acid at position 101 is Gly; the amino acid atposition 103 is Asn; the amino acid at position 28 is Ile; the aminoacid at position 30 and position 58 is Thr; the amino acid at position32, position 33, and position 35 is Tyr; the amino acid at position 50and position 62 is Glu; the amino acid at position 52, position 55,position 59, and position 61 is Asn; the amino acid at position 53 isPro; the amino acid at position 60 and position 64 is Phe; the aminoacid at position 63 and position 65 is Lys; the amino acid at position66, position 99, and position 107 is Ser; the amino acid at position100, position 104, and position 106 is Asp; the amino acid at position102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 and position 54 is Ser; the amino acid at position57 is Asp; the amino acid at position 101 is Phe; the amino acid atposition 103 is Asn; the amino acid at position 28 is Ile; the aminoacid at position 30 and position 58 is Thr; the amino acid at position32, position 33, and position 35 is Tyr; the amino acid at position 50and position 62 is Glu; the amino acid at position 52, position 55,position 59, and position 61 is Asn; the amino acid at position 53 isPro; the amino acid at position 60 and position 64 is Phe; the aminoacid at position 63 and position 65 is Lys; the amino acid at position66, position 99, and position 107 is Ser; the amino acid at position100, position 104, and position 106 is Asp; the amino acid at position102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 and position 54 is Ser; the amino acid at position57 is Asp; the amino acid at position 101 is Leu; the amino acid atposition 103 is Asn; the amino acid at position 28 is Ile; the aminoacid at position 30 and position 58 is Thr; the amino acid at position32, position 33, and position 35 is Tyr; the amino acid at position 50and position 62 is Glu; the amino acid at position 52, position 55,position 59, and position 61 is Asn; the amino acid at position 53 isPro; the amino acid at position 60 and position 64 is Phe; the aminoacid at position 63 and position 65 is Lys; the amino acid at position66, position 99, and position 107 is Ser; the amino acid at position100, position 104, and position 106 is Asp; the amino acid at position102 is Arg; and the amino acid at position 105 is Met.

In some embodiments, the peptide comprises SEQ ID NO:6 wherein the aminoacid at position 31 and position 54 is Ser; the amino acid at position57 is Asp; the amino acid at position 101 is Gly; the amino acid atposition 103 is Tyr; the amino acid at position 28 is Ile; the aminoacid at position 30 and position 58 is Thr; the amino acid at position32, position 33, and position 35 is Tyr; the amino acid at position 50and position 62 is Glu; the amino acid at position 52, position 55,position 59, and position 61 is Asn; the amino acid at position 53 isPro; the amino acid at position 60 and position 64 is Phe; the aminoacid at position 63 and position 65 is Lys; the amino acid at position66, position 99, and position 107 is Ser; the amino acid at position100, position 104, and position 106 is Asp; the amino acid at position102 is Arg; and the amino acid at position 105 is Met.

Identity or similarity with respect to this sequence is defined hereinas the percentage of amino acid residues in the modified sequence thatare identical (i.e., same residue) with the wild type 5c8 antibodyresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Typically,N-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the antibody sequence outside of the variable domain are notconstrued as affecting sequence identity or similarity. The antibody cancomprise one or more amino acid alterations in or adjacent to one ormore hypervariable regions thereof. Percent sequence identity can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

The present invention also provides nucleic acid molecules encoding anyof the peptides, or fragments thereof, described herein. A nucleic acidmolecule encoding the peptide of SEQ ID NO:3 is set forth in SEQ IDNO:2. A nucleic acid molecule encoding the peptide of SEQ ID NO:6 is setforth in SEQ ID NO:5. One skilled in the art having the amino acidsequences of the peptides disclosed herein can design and preparenucleic acid molecules encoding the peptides.

The present invention also provides vectors comprising any nucleic acidmolecule that encodes any of the peptides, or fragments thereof,described herein. One skilled in the art is readily familiar withnumerous vectors, many of which are commercially available.

The present invention also provides host cells comprising any of thenucleic acid molecules or vectors disclosed herein. The host cells canbe used, for example, to express the peptides and antibodies, orfragments of thereof. The peptides and antibodies, or fragments thereof,can also be expressed in cells in vivo. The host cell that istransformed (for example, transfected) to produce the peptides andantibodies, or fragments of thereof can be an immortalised mammaliancell line, such as those of lymphoid origin (for example, a myeloma,hybridoma, trioma or quadroma cell line). The host cell can also includenormal lymphoid cells, such as B-cells, that have been immortalized bytransformation with a virus (for example, the Epstein-Barr virus).

Some immortalized lymphoid cell lines, such as myeloma cell lines, intheir normal state, secrete isolated Ig light or heavy chains. If such acell line is transformed with a vector that expresses a mutant antibody,or fragment thereof, prepared during the process of the invention, itmay not be necessary to carry out the remaining steps of the process,provided that the normally secreted chain is complementary to thevariable domain of the Ig chain encoded by the vector prepared earlier.If the immortalised cell line, however, does not secrete or does notsecrete a complementary chain, it will be necessary to introduce intothe cells a vector that encodes the appropriate complementary chain orfragment thereof.

In the case where the immortalised cell line secretes a complementarylight or heavy chain, the transformed cell line may be produced forexample by transforming a suitable bacterial cell with the vector andthen fusing the bacterial cell with the immortalised cell line (forexample, by spheroplast fusion). Alternatively, the DNA may be directlyintroduced into the immortalised cell line by electroporation.

In some embodiments of this invention, the host cells include, but arenot limited to: bacterial cells, such as E. coli, Caulobactercrescentus, Streptomyces species, and Salmonella typhimurium; yeastcells, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pichia pastoris, Pichia methanolica; insect cell lines, such as thosefrom Spodoptera frugiperda (for example, Sf9 and Sf21 cell lines, andexpresSF™ cells (Protein Sciences Corp., Meriden, Conn., USA)),Drosophila S2 cells, and Trichoplusia in High Five® Cells (Invitrogen,Carlsbad, Calif., USA); and mammalian cells, such as COS1 and COS7cells, Chinese hamster ovary (CHO) cells, NS0 myeloma cells, NIH 3T3cells, 293 cells, HEPG2 cells, HeLa cells, L cells, HeLa, MDCK, HEK293,WI38, murine ES cell lines (for example, from strains 129/SV, C57/BL6,DBA-1, 129/SVJ), K562, Jurkat cells, and BW5147. Other useful mammaliancell lines are well known and readily available from the American TypeCulture Collection (“ATCC”) (Manassas, Va., USA) and the NationalInstitute of General Medical Sciences (NIGMS) Human Genetic CellRepository at the Coriell Cell Repositories (Camden, N.J., USA). Thesecell types are only representative and are not meant to be an exhaustivelist.

The peptides or antibodies, and fragments thereof, can be produced inprokaryotic and eukaryotic cells. The invention, thus, also providescells that express the antibodies of the present invention, includinghybridoma cells, B cells, plasma cells, as well as host cellsrecombinantly modified to express the antibodies of the presentinvention.

Among other considerations, some of which are described above, a hostcell strain may be chosen for its ability to process the expressedpeptide or antibody, or fragment thereof, in the desired fashion.Post-translational modifications of the polypeptide include, but are notlimited to, glycosylation, acetylation, carboxylation, phosphorylation,lipidation, and acylation, and it is an aspect of the present inventionto provide antibodies or fragments thereof with one or more of thesepost-translational modifications.

The present invention also provides antibodies, or fragments thereof,comprising any peptide described herein, wherein the antibody orfragment thereof can bind to human CD154. In some embodiments, theantibody, or fragment thereof, comprises any of the light chainpeptides, or fragments thereof, described herein. In other embodiments,the antibody, or fragment thereof, comprises any of the heavy chainpeptides, or fragments thereof, described herein. In yet otherembodiments, the antibody, or fragment thereof, comprises any of theheavy chain peptides, or fragments thereof, described herein and any ofthe light chain peptides, or fragments thereof, described herein.

In some embodiments, the antibody, or fragment thereof, comprises afirst peptide which is a peptide of Mutant #14, and a second peptidewhich is a peptide of mutant #1. Such a composite mutant is referred toas Mutant #16 herein.

In some embodiments, the antibody, or fragment thereof, comprises afirst peptide which is a peptide of Mutant #15, and a second peptidewhich is a peptide of mutant #2. Such a composite mutant is referred toas Mutant #17 herein.

In some embodiments, the antibody, or fragment thereof, comprises afirst peptide which is a peptide of Mutant #15, and a second peptidewhich is a peptide of mutant #1. Such a composite mutant is referred toas Mutant #18 herein.

In some embodiments, the antibody, or fragment thereof, comprises afirst peptide which is a peptide of Mutant #14, and a second peptidewhich is a peptide of mutant #4. Such a composite mutant is referred toas Mutant #19 herein.

In some embodiments, the antibody, or fragment thereof, comprises afirst peptide which is a peptide of Mutant #15, and a second peptidewhich is a peptide of mutant #4. Such a composite mutant is referred toas Mutant #20 herein.

In some embodiments, the antibody, or fragment thereof, comprises afirst peptide which is a peptide of Mutant #14, and a second peptidewhich is a peptide of mutant #2. Such a composite mutant is referred toas Mutant #23 herein.

In some embodiments, the antibody, or fragment thereof, comprises afirst peptide which is a peptide of Mutant #14, and a second peptidethat comprises SEQ ID NO:6 wherein the amino acid at position 31,position 66, position 99, and position 107 is Ser; the amino acid atposition 57, position 100, position 104, and position 106 is Asp; theamino acid at position 101 is Gly; the amino acid at position 103,position 52, position 54, position 55, position 59, and position 61 isAsn; the amino acid at position 28 is Ile; the amino acid at position 58is Thr; the amino acid at position 32, and position 35 is Tyr; the aminoacid at position 50 and position 62 is Glu; the amino acid at position53 is Pro; the amino acid at position 60 and position 64 is Phe; theamino acid at position 63 and position 65 is Lys; the amino acid atposition 102 is Arg; the amino acid at position 30 is His; the aminoacid at position 33 is Trp; and the amino acid at position 105 is Met.Such a composite mutant is referred to as Mutant #21 herein.

In some embodiments, the antibody, or fragment thereof, comprises afirst peptide which is a peptide of Mutant #15, and a second peptidethat comprises SEQ ID NO:6 wherein the amino acid at position 31,position 66, position 99, and position 107 is Ser; the amino acid atposition 57, position 100, position 104, and position 106 is Asp; theamino acid at position 101 is Gly; the amino acid at position 103,position 52, position 54, position 55, position 59, and position 61 isAsn; the amino acid at position 28 is Ile; the amino acid at position 58is Thr; the amino acid at position 32, and position 35 is Tyr; the aminoacid at position 50 and position 62 is Glu; the amino acid at position53 is Pro; the amino acid at position 60 and position 64 is Phe; theamino acid at position 63 and position 65 is Lys; the amino acid atposition 102 is Arg; the amino acid at position 30 is His; the aminoacid at position 33 is Trp; and the amino acid at position 105 is Met.Such a composite mutant is referred to as Mutant #22 herein.

In some embodiments, the antibody, or fragment thereof, comprises afirst peptide which is a peptide of Mutant #14, and a second peptidethat comprises SEQ ID NO:6 wherein the amino acid at position 31,position 54, position 66, position 99, and position 107 is Ser; theamino acid at position 57, position 100, position 104, and position 106is Asp; the amino acid at position 101 is Gly; the amino acid atposition 103, position 52, position 55, position 59, and position 61 isAsn; the amino acid at position 28 is Ile; the amino acid at position 58is Thr; the amino acid at position 32, and position 35 is Tyr; the aminoacid at position 50 and position 62 is Glu; the amino acid at position53 is Pro; the amino acid at position 60 and position 64 is Phe; theamino acid at position 63 and position 65 is Lys; the amino acid atposition 102 is Arg; the amino acid at position 30 is His; the aminoacid at position 33 is Trp; and the amino acid at position 105 is Met.Such a composite mutant is referred to as Mutant #24 herein.

In some embodiments, the antibody, or fragment thereof, comprises afirst peptide which is a peptide of Mutant #15, and a second peptidethat comprises SEQ ID NO:6 wherein the amino acid at position 31,position 54, position 66, position 99, and position 107 is Ser; theamino acid at position 57, position 100, position 104, and position 106is Asp; the amino acid at position 101 is Gly; the amino acid atposition 103, position 52, position 55, position 59, and position 61 isAsn; the amino acid at position 28 is Ile; the amino acid at position 58is Thr; the amino acid at position 32, and position 35 is Tyr; the aminoacid at position 50 and position 62 is Glu; the amino acid at position53 is Pro; the amino acid at position 60 and position 64 is Phe; theamino acid at position 63 and position 65 is Lys; the amino acid atposition 102 is Arg; the amino acid at position 30 is His; the aminoacid at position 33 is Trp; and the amino acid at position 105 is Met.Such a composite mutant is referred to as Mutant #25 herein.

In some embodiments, the antibody, or fragment thereof, comprises afirst peptide which is a peptide of Mutant #15, and a second peptidethat comprises SEQ ID NO:6 wherein the amino acid at position 31,position 66, position 99, and position 107 is Ser; the amino acid atposition 57, position 100, position 104, and position 106 is Asp; theamino acid at position 101 is Gly; the amino acid at position 103,position 52, position 54, position 55, position 59, and position 61 isAsn; the amino acid at position 28 is Ile; the amino acid at position 58is Thr; the amino acid at position 32, position 33, and position 35 isTyr; the amino acid at position 50 and position 62 is Glu; the aminoacid at position 53 is Pro; the amino acid at position 60 and position64 is Phe; the amino acid at position 63 and position 65 is Lys; theamino acid at position 102 is Arg; the amino acid at position 30 is His;and the amino acid at position 105 is Met. Such a composite mutant isreferred to as Mutant #26 herein.

In some embodiments, the antibody, or fragment thereof, comprises afirst peptide which is a peptide of Mutant #15, and a second peptidethat comprises SEQ ID NO:6 wherein the amino acid at position 31,position 66, position 99, and position 107 is Ser; the amino acid atposition 57, position 100, position 104, and position 106 is Asp; theamino acid at position 101 is Gly; the amino acid at position 103,position 52, position 54, position 55, position 59, and position 61 isAsn; the amino acid at position 28 is Ile; the amino acid at position 30and position 58 is Thr; the amino acid at position 32, and position 35is Tyr; the amino acid at position 50 and position 62 is Glu; the aminoacid at position 53 is Pro; the amino acid at position 60 and position64 is Phe; the amino acid at position 63 and position 65 is Lys; theamino acid at position 102 is Arg; the position at amino acid 33 is Trp;and the amino acid at position 105 is Met. Such a composite mutant isreferred to as Mutant #27 herein.

In some embodiments, the antibody, or fragment thereof, comprises afirst peptide which is a peptide of Mutant #14, and a second peptidethat comprises SEQ ID NO:6 wherein the amino acid at position 31,position 66, position 99, and position 107 is Ser; the amino acid atposition 57, position 100, position 104, and position 106 is Asp; theamino acid at position 101 is Gly; the amino acid at position 103,position 52, position 54, position 55, position 59, and position 61 isAsn; the amino acid at position 28 is Ile; the amino acid at position 30and position 58 is Thr; the amino acid at position 32, and position 35is Tyr; the amino acid at position 50 and position 62 is Glu; the aminoacid at position 53 is Pro; the amino acid at position 60 and position64 is Phe; the amino acid at position 63 and position 65 is Lys; theamino acid at position 102 is Arg; the position at amino acid 33 is Trp;and the amino acid at position 105 is Met. Such a composite mutant isreferred to as Mutant #28 herein.

An antibody is a glycoprotein of approximate MW 150 kD that is producedby the humoral arm of the immune system of vertebrates in response tothe presence of foreign molecules in the body. A functional antibody orantibody derivative is able to recognize and bind to its specificantigen in vitro or in vivo, and may initiate any subsequent actionsassociated with antibody-binding, including for example, directcytotoxicity, complement-dependent cytotoxicity (“CDC”),antibody-dependent cytotoxicity (“ADCC”), and antibody production.

Upon binding to the antigen, antibodies activate one or more of the manyeffector systems of the immune system that contribute to theneutralization, destruction and elimination of the infectingmicroorganism or other antigen-containing entity, for example, a cancercell.

The antibodies, and fragments thereof, disclosed herein have increasedor decreased affinity, avidity, and/or specificity as compared to thewild type 5c8 antibody. In some embodiments, the antibodies, andfragments thereof, disclosed herein have increased affinity, avidity,and/or specificity as compared to the wild type 5c8 antibody. Affinity,avidity, and/or specificity can be measured in a variety of ways.Generally, and regardless of the precise manner in which affinity isdefined or measured, the methods of the invention improve antibodyaffinity when they generate an antibody that is superior in any aspectof its clinical application to the antibody (or antibodies) from whichit was made (for example, the methods of the invention are consideredeffective or successful when a modified antibody can be administered ata lower dose or less frequently or by a more convenient route ofadministration than an antibody (or antibodies) from which it was made).

Those of ordinary skill in the art will recognize that determiningaffinity is not always as simple as looking at a single, bottom-linefigure. Since antibodies have two arms, their apparent affinity isusually much higher than the intrinsic affinity between the variableregion and the antigen. Intrinsic affinity can be measured using scFv orFab fragments.

In some embodiments, the antibody fragment is a single chain antibody(scFv), a F(ab′)₂ fragment, a Fab fragment, a Fab′ fragment, or an Fdfragment, or antigen-binding fragments thereof. A single chain antibodyis made up of variable regions linked by protein spacers in a singleprotein chain. In other embodiments, the antibody fragments includeheteromeric antibody complexes and antibody fusions, such as bispecificantibodies, hemidimeric antibodies, and multivalent antibodies (such astetravalent antibodies). A hemidimeric antibody is made up of an Fcportion and one Fab portion.

In some embodiments, the antibodies, or fragments thereof, can alsoinclude proteins containing one or more immunoglobulin light chainsand/or heavy chains, such as monomers and homo- or hetero-multimers (forexample, dimers or trimers) of these chains, where these chains areoptionally disulfide-bonded or otherwise cross-linked. These antibodies,or fragments thereof, can bind to one or more antigens.

The fragments of antibodies disclosed herein retain their ability tobind CD154. For example, for a fragment of 5c8 that is able to bind toCD154, the corresponding fragment which comprises any mutation disclosedherein is also able to bind CD154 Amino acids present on CD154 that arecontacted by 5c8 include: V126, I127, S128, E129, A130, S131, S132,K133, T134, Q139, W140, A141, E142, K143, G144, Y145, Y146, T147, M148,S149, N150, N151, V153, T154, L155, E156, N157, G158, K159, T176, F177,C178, S179, N180, E182, A183, S184, R200, F201, E202, R203, I204, L205,A209, A215, K216, P217, C218, G219, Q220, Q221, D243, P244, S245, Q246,V247, S248, H249, G250, T251, G252, F253, and T254. In some embodiments,some or all of these residues on CD154 interact with the mutantsdescribed herein. In some embodiments, the mutants described herein maycontact one or more less residues of CD154 than the wild type antibody.

In some embodiments, the antibody, or fragment thereof, is labeled witha detectable marker. Detectable markers include, but are not limited to,radioactive isotopes (such as P³² and S³⁵), enzymes (such as horseradishperoxidase, chloramphenicol acetyltransferase (CAT), β-galactosidase(β-gal), and the like), fluorochromes, chromophores, colloidal gold,dyes, and biotin. The labeled antibodies, or fragments thereof, can beused to carry out diagnostic procedures (many diagnostic assays rely ondetection of a protein antigen (such as PSA)) in a variety of cell ortissue types. For imaging procedures, in vitro or in vivo, the alteredantibodies produced by the methods described herein can be labeled withadditional agents, such as NMR contrasting agents, X-ray contrastingagents, or quantum dots. Methods for attaching a detectable agent topolypeptides, including antibodies or fragments thereof, are known inthe art. The antibodies can also be attached to an insoluble support(such as a bead, a glass or plastic slide, or the like).

In some embodiments, the antibody, or fragment thereof, is conjugated toa therapeutic agent which includes, but are not limited to,radioisotopes (such as ¹¹¹In or ⁹⁰Y), toxins (such as tetanus toxoid orricin), toxoids, and chemotherapeutic agents (see, U.S. Pat. No.6,307,026).

In some embodiments, the antibodies, or fragments thereof, modified bybeing conjugated to an imaging agent. Imaging agents include, forexample, a labeling moiety (such as biotin, fluorescent moieties,radioactive moieties, histidine tag or other peptide tags) for easyisolation or detection.

When administered, antibodies are often cleared rapidly from thecirculation and may therefore elicit relatively short-livedpharmacological activity. Consequently, frequent injections ofrelatively large doses of antibodies may be required to sustain thetherapeutic efficacy of the antibody treatment.

In some embodiments, the antibodies, or fragments thereof, can bemodified (such as by attachment to other moieties) to increase theintegrity and longevity of the antibody in vivo. For example, theantibodies, or fragments thereof, can be modified to include a moietythat can increase stabilization, thereby prolonging the serum half-lifeof the antibody.

In some embodiments, the antibody, or fragment thereof, comprises atleast one high molecular-weight polymer which include, but are notlimited to, polyethyleneimine and polylysine. In other embodiments, theantibodies, or fragments thereof, are modified by the covalentattachment of water-soluble polymers, such as polyethylene glycol,copolymers of polyethylene glycol and polypropylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinylpyrrolidone or polyproline, all of which are known to exhibitsubstantially longer half-lives in blood following intravenous injectionthan do the corresponding unmodified proteins (Abuchowski et al., In:“Enzymes as Drugs”, 1981; Holcenberg et al., Ed., 1981,Wiley-Interscience, New York, N.Y., 367-383; Anderson, Human GeneTherapy. Science, 1992, 256, 808-813; Newmark et al., J. Appl. Biochem.,1982, 4, 185-189; and Katre et al., Proc. Natl. Acad. Sci. USA, 1987,84, 1487-1491).

In some embodiments, the antibody, or fragment thereof, is PEGylated orglycosylated in at least one amino acid position or modified by othersuitable water-soluble polymers including, but are not limited to, PEG,copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose,dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly 1,3 dioxolane,poly 1,3,6 trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(nvinyl pyrrolidone) PEG, propropylene glycol homopolymers, polypropyleneoxide/ethylene oxide co polymers, polyoxyethylated polyols (e.g.,glycerol), polyvinyl alcohol, and mixtures thereof.

The polymer may be of any suitable molecular weight, and may be branchedor unbranched.

For PEG, suitable average molecular weight is between about 2 kDa andabout 100 kDa. This provides for ease in handling and manufacturing.Those of skill in the art will appreciate that in preparations of PEG,some molecules will weigh more, some less, than the stated molecularweight. Thus, molecular weight is typically specified as “averagemolecular weight.” Other molecular weights (sizes) may be used,depending on the desired therapeutic profile (e.g., the duration ofsustained release desired; the effects, if any, on biological activity;the ease in handling; the degree or lack of antigenicity and other knowneffects of PEG on a therapeutic protein). In various embodiments, themolecular weight is about 2 kDa, about 5 kDa, about 10 kDa, about 15kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 40 kDa or about 100kDa. In some embodiments, the average molecular weight of each PEG chainis about 20 kDa. In other embodiments, the average molecular weight isabout 10 kDa.

The number of polymer molecules so attached may vary, and one skilled inthe art will be able to ascertain the effect on function. One maymono-derivatize, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of PEGs). Theproportion of polymer molecules to protein (or polypeptide) moleculeswill vary, as will their concentrations in the reaction mixture. Ingeneral, the optimum ratio (in terms of efficiency of reaction in thatthere is no excess unreacted protein or polymer) will be determined byfactors such as the desired degree of derivatization (e.g., mono, di-,tri-, etc.), the molecular weight of the polymer selected, whether thepolymer is branched or unbranched, and the reaction conditions.

The PEG molecules (or other chemical moieties) should be attached to theprotein with consideration of effects on functional or antigenic domainsof the protein. There are a number of attachment methods available tothose skilled in the art. See, for example, EP 0 401384 (coupling PEG toG CSF); Malik et al., Exp. Hematol., 1992, 20, 1028-1035 (reportingPEGylation of GM CSF using tresyl chloride).

For example, PEG may be covalently bound (PEGylation) through amino acidresidues via a reactive group, such as, a free amino or carboxyl group.The amino acid residues having a free amino group include lysineresidues and the amino-terminal amino acid residue. Those having a freecarboxyl group include aspartic acid residues, glutamic acid residues,and the C terminal amino acid residue. Sulfhydryl groups may also beused as a reactive group for attaching the PEG molecule(s). Fortherapeutic purposes, attachment can be at an amino group, for example,at the N terminus or lysine group. One may specifically desire anamino-terminal chemically modified protein.

Using PEG as an illustration of the present compositions, one may selectfrom a variety of PEG molecules (by molecular weight, branching, etc.),the proportion of PEG molecules to protein (or peptide) molecules in thereaction mix, the type of PEGylation reaction to be performed, and themethod of obtaining the selected amino-terminally PEGylated protein. Themethod of obtaining the amino-terminal PEGylated preparation (i.e.,separating this moiety from other monoPEGylated moieties if necessary)may be by purification of the amino-terminal PEGylated material from apopulation of PEGylated protein molecules. Selective amino-terminalchemical modification may be accomplished by reductive alkylation thatexploits differential reactivity of different types of primary aminogroups (lysine versus the amino-terminal) available for derivatizationin a particular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at theamino-terminus with a carbonyl group containing polymer is achieved. Forexample, one may selectively PEGylate the amino-terminus of the proteinby performing the reaction at a pH which allows one to take advantage ofthe pKa differences between the epsilon (∈) amino group of the lysineresidues and that of the alpha (α) amino group of the amino-terminalresidue of the protein. By such selective derivatization, attachment ofa water soluble polymer to a protein is controlled: the conjugation withthe polymer takes place predominantly at the amino terminus of theprotein and no significant modification of other reactive groups, suchas the lysine side chain amino groups, occurs.

Using reductive alkylation, the water-soluble polymer may be of the typedescribed above, and should have a single reactive aldehyde for couplingto the protein. PEG propionaldehyde, containing a single reactivealdehyde, may be used.

Antibody modifications can also increase the protein's solubility inaqueous solution, eliminate aggregation, enhance the physical andchemical stability of the protein, and greatly reduce the immunogenicityand antigenicity of the protein. As a result, the desired in vivobiological activity can be achieved by the administration of suchpolymer-protein adducts less frequently or in lower doses than with theunmodified protein.

The term “chimeric antibody” is used to describe a protein comprising atleast an antigen-binding portion of an immunoglobulin molecule that isattached by, for example, a peptide bond or peptide linker, to aheterologous protein or a peptide thereof. The “heterologous” proteincan be a non-immunoglobulin or a portion of an immunoglobulin of adifferent species, class or subclass.

There are numerous processes by which such antibodies can be made. Forexample, one can prepare an expression vector including a promoter thatis operably linked to a DNA sequence that encodes at least V_(H) orV_(L) and a sequence that encodes the heterologous protein (or a peptidethereof (the peptide being of a sufficient length that it can berecognized as a non-immunoglobulin molecule (for example, a peptidehaving no substantial sequence identity to an immunoglobulin))). Ifnecessary, or desired, one can prepare a second expression vectorincluding a promoter that is operably linked to a DNA sequence thatencodes the complementary variable domain (for example, where the parentexpression vector encodes V_(H), the second expression vector encodesV_(L) and vice versa). A cell line (for example, an immortalizedmammalian cell line) can then be transformed with one or both of theexpression vectors and cultured under conditions that permit expressionof the chimeric variable domain or chimeric antibody (see, for example,International Patent Application No. PCT/GB85/00392). This method can beused to express the modified antibodies of the present invention,antibodies containing full-length heavy and light chains, or fragmentsthereof (for example, the Fab, F(ab′)₂, or scFv fragments describedherein). The methods are not limited to expression of chimericantibodies.

Though naturally occurring antibodies are derived from a single species,engineered antibodies and antibody fragments may be derived from morethan one species of animal,—for example, chimeric antibodies. To date,mouse (murine)/human chimeric and murine/non-human primate antibodieshave been generated, though other species' combinations are possible.

In some embodiments, the antibodies, or fragments thereof, are chimericantibodies. Typically, chimeric antibodies include the heavy and/orlight chain variable regions, including both complementary determiningregion (“CDR”) and framework residues, of one species, (typically mouse)fused to constant regions of another species (typically human). Thesechimeric mouse/human antibodies contain approximately 75% human and 25%mouse amino acid sequences, respectively. The human sequences representthe constant regions of the antibody, while the mouse sequencesrepresent the variable regions (and thus contain the antigen-bindingsites) of the antibody.

The rationale for using such chimeras is to retain the antigenspecificity of the mouse antibody but reduce the immunogenicity of themouse antibody (a mouse antibody would cause an immune response againstit in species other than the mouse) and thus be able to employ thechimera in human therapies.

In another embodiment, the antibodies, or fragments thereof, includechimeric antibodies comprising framework regions from one antibody andCDR regions from another antibody. In other embodiments, the antibodies,or fragments thereof, include chimeric antibodies comprising CDR regionsfrom different human antibodies. In other embodiments, the antibodies,or fragments thereof, include chimeric antibodies comprising CDR regionsfrom at least two different human antibodies.

Methods of making all of the chimeric antibodies described above arewell known to one of skill in the art (U.S. Pat. No. 5,807,715; Morrisonet al., Proc. Natl. Acad. Sci. USA, 1984, 81, 6851-5; Sharon et al.,Nature, 1984, 309, 364-7; and Takeda et al., Nature, 1985 314, 452-4).

In another embodiment, the antibodies, or fragments thereof, alsoinclude primatized, humanized and fully human antibodies. Primatized andhumanized antibodies typically include heavy and/or light chain CDRsfrom a murine antibody grafted into a non-human primate or humanantibody V region framework, usually further comprising a human constantregion (Riechmann et al., Nature, 1988, 332, 323-7; Co et al., Nature,1991, 351, 501-2; and U.S. Pat. Nos. 6,054,297, 5,821,337, 5,770,196,5,766,886, 5,821,123, 5,869,619, 6,180,377, 6,013,256, 5,693,761, and6,180,370).

A humanized antibody is an antibody produced by recombinant DNAtechnology, in which some or all of the amino acids of a humanimmunoglobulin light or heavy chain that are not required for antigenbinding (for example, the constant regions and the framework regions ofthe variable domains) are used to substitute for the corresponding aminoacids from the light or heavy chain of the cognate, nonhuman antibody.By way of example, a humanized version of a murine antibody to a givenantigen has on both of its heavy and light chains 1) constant regions ofa human antibody; 2) framework regions from the variable domains of ahuman antibody; and 3) CDRs from the murine antibody. When necessary,one or more residues in the human framework regions can be changed toresidues at the corresponding positions in the murine antibody so as topreserve the binding affinity of the humanized antibody to the antigen.This change is sometimes called “back mutation.” Humanized antibodiesgenerally are less likely to elicit an immune response in humans ascompared to chimeric human antibodies because the former containconsiderably fewer non-human components. Methods for making humanizedantibodies are well know to those of skill in the art of antibodies(European Patent No. 239400; Jones et al., Nature, 1986, 321, 522-525;Riechmann et al., Nature, 1988, 332, 323-327; Verhoeyen et al., Science,1988, 239, 1534-1536; Queen et al., Proc. Nat. Acad. Sci. USA, 1989, 86,10029; Orlandi et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 3833; andU.S. Pat. No. 6,180,370).

In some embodiments, humanized antibodies are generated by thetransplantation of murine (or other non-human) CDRs onto a humanantibody. More specifically, this can be achieved as follows: 1) thecDNAs encoding heavy and light chain variable domains are isolated froma hybridoma; 2) the DNA sequences of the variable domains, including theCDRs, are determined by sequencing; 3) the DNAs encoding the CDRs aretransferred to the corresponding regions of a human antibody heavy orlight chain variable domain coding sequence by site directedmutagenesis; and 4) the human constant region gene segments of a desiredisotype (for example, 1 for CH and k for CL) are added. Finally, thehumanized heavy and light chain genes are co-expressed in mammalian hostcells (for example, CHO or NS0 cells) to produce soluble humanizedantibody.

At times, direct transfer of CDRs to a human framework leads to a lossof antigen-binding affinity of the resultant antibody. This is becausein some cognate antibodies, certain amino acids within the frameworkregions interact with the CDRs and thus influence the overall antigenbinding affinity of the antibody. In such cases, it would be critical tointroduce “back mutations” in the framework regions of the acceptorantibody in order to retain the antigen-binding activity of the cognateantibody. The general approaches of making back mutations is well knownto those of skill in the art (Queen et al., Proc. Nat. Acad. Sci. USA,1989, 86, 10029; and Co et al., Proc. Nat. Acad. Sci. USA, 1991, 88,2869-2873; PCT patent application WO 90/07861; and Tempest,Biotechnology, 1991, 9, 266-271).

In some embodiments, the antibodies, or fragments thereof, are fullyhuman anti-CD154 antibodies. The fully human antibodies can be preparedusing in vitro-primed human splenocytes, (Boerner et al., J. Immunol.,1991, 147, 86-95) or phage-displayed antibody libraries (U.S. Pat. No.6,300,064). Alternately, the fully human antibodies can be prepared byrepertoire cloning (Persson et al., Proc. Nat. Acad. Sci. USA, 1991, 88,2432-2436; and Huang et al., J. Immunol. Methods, 1991, 141, 227-236).In addition, U.S. Pat. No. 5,798,230 describes preparation of humanmonoclonal antibodies from human B cells, wherein humanantibody-producing B cells are immortalized by infection with anEpstein-Barr virus, or a derivative thereof, that expresses Epstein-Barrvirus nuclear antigen 2 (“EBNA2”), a protein required forimmortalization. The EBNA2 function is subsequently shut off, resultingin an increase in antibody production.

Other methods for producing fully human antibodies involve the use ofnon-human animals that have inactivated endogenous Ig loci and aretransgenic for un-rearranged human antibody heavy chain and light chaingenes. Such transgenic animals can be immunized with activated T cellsor the D1.1 protein (U.S. Pat. Nos. 5,474,771; 6,331,433; and 6,455,044)and hybridomas can be generated from B cells derived there from. Thedetails of these methods are described in the art. See, e.g. the variousGenPharm/Medarex (Palo Alto, Calif.) publications/patents concerningtransgenic mice containing human Ig miniloci, including U.S. Pat. No.5,789,650; the various Abgenix (Fremont, Calif.) publications/patentswith respect to XENOMOUSE® mice, including U.S. Pat. Nos. 6,075,181,6,150,584 and 6,162,963; Green et al., Nature Genetics, 1994, 7, 13-21;Mendez et al., Nature Genetics, 1997, 15, 146-56; and the various Kirin(Japan) publications/patents concerning “transomic” mice, includingEuropean Patent 843961 and Tomizuka et al., Nature Genetics, 1997 June;16(2):133-43.

Once the sequence of an antibody (for example, a CDR-grafted orotherwise modified or “humanized” antibody) has been decided upon, thatantibody can be made by techniques well known in the art of molecularbiology. More specifically, recombinant DNA techniques can be used toproduce a wide range of polypeptides by transforming a host cell with anucleic acid sequence (for example, a DNA sequence that encodes thedesired protein products (for example, a modified heavy or light chain;the variable domains thereof, or other antigen-binding fragmentsthereof)).

More specifically, the methods of production can be carried out asdescribed above for chimeric antibodies. The DNA sequence encoding, forexample, an altered variable domain can be prepared by oligonucleotidesynthesis. The variable domain can be one that includes the FRs of ahuman acceptor molecule and the CDRs of a donor, for example, murine,either before or after one or more of the residues (for example, aresidue within a CDR) has been modified to facilitate antigen binding.This is facilitated by determining the framework region sequence of theacceptor antibody and at least the CDR sequences of the donor antibody.Alternatively, the DNA sequence encoding the altered variable domain maybe prepared by primer directed oligonucleotide site-directedmutagenesis. This technique involves hybridizing an oligonucleotidecoding for a desired mutation with a single strand of DNA containing themutation point and using the single strand as a template for extensionof the oligonucleotide to produce a strand containing the mutation. Thistechnique, in various forms, is described by, for example, Zoller andSmith (Nuc. Acids Res., 1982, 10, 6487-6500), Norris et al. (Nuc. AcidsRes., 1983, 11, 5103-5112), Zoller and Smith (DNA, 1984, 3, 479-488),and Kramer et al. (Nuc. Acids Res., 1982, 10, 6475-6485).

Other methods of introducing mutations into a sequence are known as welland can be used to generate the altered antibodies described herein(see, for example, Carter et al., Nuc. Acids Res., 1985, 13, 4431-4443).The oligonucleotides used for site-directed mutagenesis can be preparedby oligonucleotide synthesis or isolated from DNA coding for thevariable domain of the donor antibody by use of suitable restrictionenzymes.

In another embodiment, the antibodies, or fragments thereof, can beprepared by cell free translation. Alternately, the antibodies, orfragments thereof, can be produced in bioreactors containing theantibody-expressing cells, in order to facilitate large scaleproduction.

In some embodiments, the antibodies, or fragments thereof, can beproduced in transgenic mammals, such as goats, cows, or sheep, thatexpress the antibody in milk, in order to facilitate large scaleproduction of antibodies (U.S. Pat. No. 5,827,690; and Pollock et al.,J. Immunol. Meth., 1999, 231, 147-57).

The present invention also provides compositions comprising any one ormore of the peptides, or fragments thereof, any one or more of thenucleic acid molecules or vectors, or any one or more of the antibodies,or fragments thereof, described herein. Compositions include, forexample, pharmaceutical compositions.

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a subject suffering from a disorder in an amountsufficient to eliminate or reduce the risk, lessen the severity, ordelay the outset of the disorder, including biochemical, histologicand/or behavioral symptoms of the disorder, its complications andintermediate pathological phenotypes presenting during development ofthe disorder. In therapeutic applications, compositions or medicamentsare administered to a subject suspected of, or already suffering fromsuch a disorder in an amount sufficient to cure, or at least partiallyarrest, the symptoms of the disorder (biochemical, histologic and/orbehavioral), including its complications and intermediate pathologicalphenotypes in development of the disorder.

The pharmaceutical compositions of the invention can include at leastone antibody, or fragment thereof, disclosed herein in apharmaceutically acceptable carrier. A “pharmaceutically acceptablecarrier” refers to at least one component of a pharmaceuticalpreparation that is normally used for administration of activeingredients. As such, a carrier can contain any pharmaceutical excipientused in the art and any form of vehicle for administration. Carriersinclude, but are not limited to, phosphate buffered saline,physiological saline, water, citrate/sucrose/Tween formulations andemulsions such as, for example, oil/water emulsions.

The compositions can include an active therapeutic agent and a varietyof other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.(1980)). The desired form depends on the intended mode of administrationand therapeutic application. The compositions can also include,depending on the formulation desired, pharmaceutically acceptable,non-toxic carriers or diluents, which are defined as vehicles commonlyused to formulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the combination. Examples of such diluentsinclude, but are not limited to, distilled water, physiologicalphosphate-buffered saline, Ringer's solutions, dextrose solution, andHank's solution. In addition, the pharmaceutical composition orformulation may also include other carriers, adjuvants, or nontoxic,nontherapeutic, nonimmunogenic stabilizers and the like. Solidformulations of the compositions for oral administration can containsuitable carriers or excipients, such as corn starch, gelatin, lactose,acacia, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalciumphosphate, calcium carbonate, sodium chloride, or alginic acid.Disintegrators that can be used include, without limitation,microcrystalline cellulose, corn starch, sodium starch glycolate, andalginic acid. Tablet binders that can be used include acacia,methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone(Povidone™), hydroxypropyl methylcellulose, sucrose, starch, andethylcellulose. Lubricants that can be used include magnesium stearates,stearic acid, silicone fluid, talc, waxes, oils, and colloidal silica.Additional excipients include, for example, colorants, taste-maskingagents, solubility aids, suspension agents, compressing agents, entericcoatings, sustained release aids, and the like.

Antibodies can be administered in the form of a depot injection orimplant preparation, which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises antibody, or fragment thereof, at 5 mg/ml,formulated in aqueous buffer consisting of 50 mM L-histidine and 150 mMNaCl, adjusted to pH 6.0 with HCl. Another example of a suitableformulation buffer for monoclonal antibodies contains 20 mM sodiumcitrate, pH 6.0, 10% sucrose, and 0.1% Tween 80.

In some embodiments, liquid formulations of a pharmaceutical compositionfor oral administration prepared in water or other aqueous vehicles cancontain various suspending agents such as methylcellulose, alginates,tragacanth, pectin, kelgin, carrageenan, acacia, polyvinylpyrrolidone,and polyvinyl alcohol. Liquid formulations of pharmaceuticalcompositions of this invention can also include solutions, emulsions,syrups and elixirs containing, together with the active compound(s),wetting agents, sweeteners, and coloring and flavoring agents. Variousliquid and powder formulations of the pharmaceutical compositions can beprepared by conventional methods for inhalation into the lungs of themammal to be treated.

In some embodiments, liquid formulations of a pharmaceutical compositionfor injection can comprise various carriers such as vegetable oils,dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate,isopropyl myristate, ethanol, polyols such as, for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like. In someembodiments, the composition includes a citrate/sucrose/tween carrier.For intravenous injections, water soluble versions of the compositionscan be administered by the drip method, whereby a pharmaceuticalformulation containing the antifungal agent and a physiologicallyacceptable excipient is infused. Physiologically acceptable excipientscan include, for example, 5% dextrose, 0.9% saline, Ringer's solution orother suitable excipients. A suitable insoluble form of the compositioncan be prepared and administered as a suspension in an aqueous base or apharmaceutically acceptable oil base, such as an ester of a long chainfatty acid such as, for example, ethyl oleate.

The compositions can be, for example, injectable solutions, aqueoussuspensions or solutions, non-aqueous suspensions or solutions, solidand liquid oral formulations, salves, gels, ointments, intradermalpatches, creams, lotions, tablets, capsules, sustained releaseformulations, and the like. In some embodiments, for topicalapplications, the pharmaceutical compositions can be formulated in asuitable ointment. In some embodiments, a topical semi-solid ointmentformulation typically comprises a concentration of the active ingredientfrom about 1 to 20%, or from 5 to 10%, in a carrier, such as apharmaceutical cream base. Some examples of formulations of acomposition for topical use include, but are not limited to, drops,tinctures, lotions, creams, solutions, and ointments containing theactive ingredient and various supports and vehicles.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicroparticles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect (see Langer, Science, 1990, 249, 1527 andHanes, Advanced Drug Delivery Reviews, 1997, 28, 97). A sterileinjectable preparation such as, for example, a sterile injectableaqueous or oleaginous suspension can also be prepared. This suspensionmay be formulated according to techniques known in the art usingsuitable dispersing, wetting, and suspending agents. In someembodiments, the pharmaceutical composition can be delivered in amicroencapsulation device so as to reduce or prevent a host immuneresponse against the protein.

Effective doses of the compositions of the present invention, for thetreatment of a condition vary depending upon many different factors,including means of administration, target site, physiological state ofthe subject, whether the subject is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the subject is a human but non-human mammalsincluding transgenic mammals can also be treated.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, from about 0.01 and 200 mg/kg, or from about 0.01to 20 mg/kg of the host body weight. For example, dosages can be 1 mg/kgbody weight or 10 mg/kg body weight or within the range of 1 to 10mg/kg, or at least 1 mg/kg. Subjects can be administered such dosesdaily, on alternative days, weekly or according to any other scheduledetermined by empirical analysis. An exemplary treatment entailsadministration in multiple dosages over a prolonged period, for example,of at least six months. Additional exemplary treatment regimes entailadministration once per every two weeks or once a month or once every 3to 6 months. Exemplary dosage schedules include 1 to 10 mg/kg or 15mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kgweekly. In some embodiments, two or more antibodies, or fragmentsthereof, with different binding specificities are administeredsimultaneously, in which case the dosage of each antibody administeredfalls within the ranges indicated.

Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. In someembodiments, dosage is adjusted to achieve a plasma antibodyconcentration of 1 to 1000 mg/ml and in some methods 25 to 300 μg/ml.Alternately, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe subject. In general, human antibodies show the longest half-life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies, in descending order.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions containing the present antibodies or acocktail thereof are administered to a subject not already in thedisease state to enhance the subject's resistance. Such an amount isdefined to be a “prophylactic effective dose.” In this use, the preciseamounts again depend upon the subject's state of health and generalimmunity, but generally range from 0.1 to 25 mg per dose, especially 0.5to 2.5 mg per dose. A relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somesubjects continue to receive treatment for the rest of their lives.

In therapeutic applications, a relatively high dosage (for example, fromabout 1 to 200 mg of antibody per dose, with dosages of from 5 to 25 mgbeing more commonly used) at relatively short intervals is sometimesrequired until progression of the disease is reduced or terminated, andpossibly until the subject shows partial or complete amelioration ofsymptoms of disease. Thereafter, the patent can be administered aprophylactic regime.

In some embodiments, the antibodies, or fragments thereof, orcompositions comprising the same, can be administered to a subject byinjection intravenously, subcutaneously, intraperitoneally,intramuscularly, intramedullarily, intraventricularly, intraepidurally,intraarterially, intravascularly, intraarticularly, intrasynovially,intrasternally, intrathecally, intrahepatically, intraspinally,intratumorly, intracranially, enteral, intrapulmonary, transmucosal,intrauterine, sublingual, or locally at sites of inflammation or tumorgrowth by using standard methods. Alternately, the antibodies, orfragments thereof, or compositions comprising the same, can beadministered to a subject by routes including oral, nasal, ophthalmic,rectal, or topical. The most typical route of administration of aprotein drug is intravascular, subcutaneous, or intramuscular, althoughother routes can be effective. In some embodiments, antibodies areadministered as a sustained release composition or device, such as aMedipad™ device. The protein drug can also be administered via therespiratory tract, for example, using a dry powder inhalation device,nebulizer, or a metered dose inhaler.

In some embodiments, the antibodies, or fragments thereof, can beadministered to a subject by sustained release administration, by suchmeans as depot injections of erodible implants directly applied duringsurgery or by implantation of an infusion pump or a biocompatiblesustained release implant into the subject. Alternately, the antibodies,or fragments thereof, can be administered to a subject by injectabledepot routes of administration, such as by using 1-, 3-, or 6-monthdepot injectable or biodegradable materials and methods, or by applyingto the skin of the subject a transdermal patch containing the antibody,antibody derivative or pharmaceutical composition, and leaving the patchin contact with the subject's skin, generally for 1 to 5 hours perpatch.

The compositions can optionally be administered in combination withother agents that are at least partly effective in treatment of immunedisorders. In some embodiments, the pharmaceutical composition furthercomprises an immunosuppressive or immunomodulatory compound. Forexample, such an immunosuppressive or immunomodulatory compound may beone of the following: an agent that interrupts T cell costimulatorysignaling via CD28; an agent that interrupts calcineurin signaling, acorticosteroid, an anti-proliferative agent, and an antibody thatspecifically binds to a protein expressed on the surface of immune cellsincluding, but not limited to, CD45, CD2, IL2R, CD4, CD8 and RANK FcR,B7, CTLA4, TNF, LTβ, and VLA-4. In addition, in some embodiments, theimmunosuppressive or immunomodulatory compound is tacrolimus, sirolimus,mycophenolate mofetil, mizorubine, deoxyspergualin, brequinar sodium,leflunomide, rapamycin or azaspirane.

The present invention also provides kits comprising any of theantibodies, or fragments thereof, described herein. The kit can include,for example, container, package or dispenser along with labels andinstructions for administration or use.

The present invention also provides methods of treating or preventing aCD154-related human disease or disorder. In some embodiments, the methodcomprises administering to a human a therapeutically- orprophylactically-effective amount of any of the antibodies, or fragmentsthereof, described herein, or a composition comprising the same, suchthat the CD154-related human disease or disorder is diminished orprevented. For the purposes of this invention, “administration” meansany of the standard methods of administering an antibody, antibodyfragment, or composition known to those skilled in the art, and shouldnot be limited to the examples provided herein.

In some embodiments, the subject being treated will have been previouslydiagnosed as having a disease or condition suitable for treatmentsdisclosed herein. Such subjects will, thus, have been diagnosed as baingin need of treatment. Alternately, the treatment may be intended totreat a particular disease or condition but simultaneously be treatinganother undiagnosed condition as well.

Treatment of a subject suffering from a disease or disorder can bemonitored using standard methods. Some methods entail determining abaseline value, for example, of an antibody level or profile in asubject, before administering a dosage of agent, and comparing this witha value for the profile or level after treatment. A significant increasesuch as, for example, greater than the typical margin of experimentalerror in repeat measurements of the same sample, expressed as onestandard deviation from the mean of such measurements in value of thelevel or profile signals a positive treatment outcome (i.e., thatadministration of the agent has achieved a desired response). If thevalue for immune response does not change significantly, or decreases, anegative treatment outcome is indicated.

In other embodiments, a control value such as a mean and standarddeviation, of level or profile is determined for a control population.Typically the individuals in the control population have not receivedprior treatment. Measured values of the level or profile in a subjectafter administering a therapeutic agent are then compared with thecontrol value. A significant increase relative to the control value,such as greater than one standard deviation from the mean, signals apositive or sufficient treatment outcome. A lack of significant increaseor a decrease signals a negative or insufficient treatment outcome.Administration of the therapeutic is generally continued while the levelis increasing relative to the control value. As before, attainment of aplateau relative to control values is an indicator that theadministration of treatment can be discontinued or reduced in dosageand/or frequency.

In other embodiments, a control value of the level or profile, such as amean and standard deviation, is determined from a control population ofindividuals who have undergone treatment with a therapeutic agent andwhose levels or profiles have plateaued in response to treatment.Measured values of levels or profiles in a subject are compared with thecontrol value. If the measured level in a subject is not significantlydifferent, such as by more than one standard deviation, from the controlvalue, treatment can be discontinued. If the level in a subject issignificantly below the control value, continued administration of agentis warranted. If the level in the subject persists below the controlvalue, then a change in treatment may be indicated.

In other embodiments, a subject who is not presently receiving treatmentbut has undergone a previous course of treatment is monitored forantibody levels or profiles to determine whether a resumption oftreatment is required. The measured level or profile in the subject canbe compared with a value previously achieved in the subject after aprevious course of treatment. A significant decrease relative to theprevious measurement, such as greater than a typical margin of error inrepeat measurements of the same sample, is an indication that treatmentcan be resumed. Alternately, the value measured in a subject can becompared with a control value (mean plus standard deviation) determinedin a population of subjects after undergoing a course of treatment.Alternately, the measured value in a subject can be compared with acontrol value in populations of prophylactically treated subjects whoremain free of symptoms of disease, or populations of therapeuticallytreated subjects who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the controllevel, such as more than a standard deviation, is an indicator thattreatment should be resumed in a subject.

The antibody profile following administration typically shows animmediate peak in antibody concentration followed by an exponentialdecay. Without a further dosage, the decay approaches pretreatmentlevels within a period of days to months depending on the half-life ofthe antibody administered. For example the half-life of some humanantibodies is of the order of 20 days.

In some methods, a baseline measurement of antibody to a given antigenin the subject is made before administration, a second measurement ismade soon thereafter to determine the peak antibody level, and one ormore further measurements are made at intervals to monitor decay ofantibody levels. When the level of antibody has declined to baseline ora predetermined percentage of the peak less baseline, such as 50%, 25%or 10%, administration of a further dosage of antibody is administered.In some embodiments, peak or subsequent measured levels less backgroundare compared with reference levels previously determined to constitute abeneficial prophylactic or therapeutic treatment regime in othersubjects. If the measured antibody level is significantly less than areference level, such as less than the mean minus one standard deviationof the reference value in population of subjects benefiting fromtreatment, administration of an additional dosage of antibody isindicated.

Additional embodiments include monitoring, over the course of treatment,any art-recognized physiologic symptom, such as physical or mentalsymptom, routinely relied on by researchers or physicians to diagnose ormonitor disorders.

In some embodiments, the antibodies, or fragments thereof, disclosedherein, or a pharmaceutical composition comprising the same, are capableof inhibiting an immune response in a subject. The antibody, antibodyderivative or pharmaceutical composition is administered to the subjectin an effective inhibiting amount. An “effective inhibiting amount” ofan antibody, antibody derivative, such as a CD154-binding fragment, orpharmaceutical composition is any amount which is effective to inhibitthe CD154-CD40 interaction in the subject to whom it is administered.Methods of determining an “inhibiting amount” are well known to thoseskilled in the art and depend upon factors including, but not limitedto, the type of subject involved, the size of the subject and thetherapeutic agent delivered.

In a particular embodiment, the anti-CD154 antibody, antibody derivativeor pharmaceutical composition comprising the antibody or antibodyderivative is capable of binding to the CD 154 molecule. In anotherembodiment, the anti-CD 154 antibody, antibody derivative orpharmaceutical composition comprising the antibody or antibodyderivative is capable of inhibiting the immune response by inhibitingthe CD154-CD40 interaction.

In some embodiments, the anti-CD154 antibody, antibody derivative orpharmaceutical composition comprising the antibody or antibodyderivative is capable of inhibiting inflammation. For the purposes ofthis invention, inflammatory responses are characterized by redness,swelling, heat and pain, as consequences of capillary dilation withedema and migration of phagocytic leukocytes. Some examples ofinflammatory responses include, but are not limited to, arthritis,contact dermatitis, hyper-IgE syndrome, inflammatory bowel disease,allergic asthma, and idiopathic inflammatory disease. Some examples ofarthritis include, but are not limited to, rheumatoid arthritis,non-rheumatoid inflammatory arthritis, arthritis associated with Lymedisease and inflammatory osteoarthritis. Some examples of idiopathicinflammatory disease include, but are not limited to, psoriasis andsystemic lupus erythematosus. Additional diseases, disorders, andconditions that can be treated using the compounds and/or compositionsdisclosed herein include, but are not limited to, atherosclerosis,Myasthenia gravis, Graves' disease, idiopathic thrombocytopenia purpura,hemolytic anemia, diabetes mellitus, Crohn's disease, multiplesclerosis, and drug-induced autoimmune diseases, such as drug-inducedlupus.

In some embodiments, the compounds and compositions can be used toinhibit rejection by the subject of a transplanted organ such as, forexample, a transplanted heart, kidney, liver, skin, pancreatic isletcells or bone marrow. In other embodiments, the compounds andcompositions can be used to inhibit graft-vs-host disease, allergicresponses such as hay fever or an allergy to penicillin or other drugs,an autoimmune response in a subject suffering from an autoimmuneresponse which is derived from an infectious disease, or inhibitingfibrosis in a subject. Examples of fibrosis include, but are not limitedto, pulmonary fibrosis or fibrotic disease. Examples of pulmonaryfibrosis include, but are not limited to, pulmonary fibrosis secondaryto adult respiratory distress syndrome, drug-induced pulmonary fibrosis,idiopathic pulmonary fibrosis, and hypersensitivity pneumonitis.Examples of fibrotic diseases include, but are not limited to,Hepatitis-C, Hepatitis-B, cirrhosis, cirrhosis of the liver secondary toa toxic insult, cirrhosis of the liver secondary to drugs, cirrhosis ofthe liver secondary to a viral infection, and cirrhosis of the liversecondary to an autoimmune disease.

In some embodiments, the compounds and compositions can inhibit anautoimmune response in a subject suffering from an autoimmune responsewhich is derived from, for example, Reiter' syndrome, spondyloarthritis,Lyme disease, HIV infection, syphilis, or tuberculosis.

In some embodiments, the compounds and compositions can inhibitgastrointestinal disease such as, for example, esophageal dysmotility,inflammatory bowel disease, and scleroderma. Alternately, the compoundsand compositions can inhibit vascular disease such as, for example,atherosclerosis and reperfusion injury.

In some embodiments, the compounds and compositions can inhibit theproliferation of T cell tumor cells in a subject suffering from a T cellcancer such as, for example, a T cell leukemia or lymphoma. Alternately,the compounds and compositions can inhibit viral infection of T cells ofa subject by the HTLV I virus.

In some embodiments, the compounds and compositions can be used forimaging tumor cells or neoplastic cells in a subject that express aprotein that is specifically recognized by hu5c8. One method for imagingtumor cells or neoplastic cells in a subject comprises: administering tothe subject an effective amount of the antibody, or fragment thereof, orcomposition comprising the same, under conditions permitting theformation of a complex between the antibody or antibody fragment and aprotein on the surface of tumor cells or neoplastic cells, and imagingany antibody/protein complex or antibody fragment/complex formed,thereby imaging any tumor cells or neoplastic cells in the subject.

In some embodiments, the compounds and compositions can be used todetect the presence of tumor cells or neoplastic cells in a subject thatexpress a protein that is specifically recognized by hu5c8. One methodfor detecting the presence of tumor cells or neoplastic cells in asubject comprises: administering to the subject an effective amount ofthe antibody, or antibody fragment, or a composition comprising thesame, under conditions permitting the formation of a complex between theantibody or antibody fragment and a protein, clearing any unboundimaging agent from the subject, and detecting the presence of anyantibody/protein complex or antibody fragment/complex formed, thepresence of such complex indicating the presence of tumor cells orneoplastic cells in the subject.

The antibodies, or fragments thereof, and compositions comprising thesame, can be administered as a single dosage for certain indications,such as preventing immune response to an antigen to which a subject isexposed for a brief time, such as an exogenous antigen administered on asingle day of treatment. Examples of such a therapy would includecoadministration of the antibody or antibody derivative of the inventionalong with a therapeutic agent, for example an antigenic pharmaceutical,an allergen or a blood product, or a gene therapy vector. In indicationswhere antigen is chronically present, such as in controlling immunereaction to transplanted tissue or to chronically administered antigenicpharmaceuticals, the antibodies, antibody derivatives or pharmaceuticalcompositions of the invention are administered at intervals for as longa time as medically indicated, ranging from days or weeks to the life ofthe subject.

In some embodiments, the subject(s) that can be treated by theabove-described methods is an animal, such as a mammal, including, butare not limited to, humans, non-human primates, rodents (including rats,mice, hamsters and guinea pigs) cow, horse, sheep, goat, pig, dog andcat. In most instances, the mammal is a human.

The present invention also provides use of any of the peptides, orfragments thereof, or antibodies, or fragments thereof, described hereinfor treating a human disease or disorder associated with CD154.

The present invention also provides use of any of the peptides, orfragments thereof, or antibodies, or fragments thereof, described hereinin the manufacture of a medicament for the treatment of a human diseaseor disorder associated with CD154.

The embodiments disclosed herein may be better understood based on theExamples that follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described more fully in the embodimentsof the invention that follow thereafter.

Throughout this application, various publications and references arereferred to within parenthesis. Disclosures of these publications andreferences, in their entireties, are hereby incorporated by referenceinto this application to more fully describe the state of the art towhich this invention pertains. The materials, methods, and examples areillustrative only and not intended to be limiting.

Standard reference works setting forth the general principles ofrecombinant DNA technology known to those of skill in the art includeAusubel et al., Current Protocols In Molecular Biology, John Wiley &Sons, New York (1998 and Supplements to 2001); Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory Press, Plainview, N.Y. (1989); Kaufman et al., Eds., HandbookOf Molecular And Cellular Methods In Biology And Medicine, CRC Press,Boca Raton (1995); McPherson, Ed., Directed Mutagenesis: A PracticalApproach, IRL Press, Oxford (1991).

Standard reference works setting forth the general principles ofimmunology known to those of skill in the art include: Harlow and Lane,Antibodies: A Laboratory Manual, 2d Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1999); and Roitt et al., Immunology, 3dEd., Mosby-Year Book Europe Limited, London (1993). Standard referenceworks setting forth the general principles of medical physiology andpharmacology known to those of skill in the art include: Fauci et al.,Eds., Harrison's Principles Of Internal Medicine, 14th Ed., McGraw-HillCompanies, Inc. (1998).

EXEMPLIFICATION

Throughout the examples, the following materials and methods were usedunless otherwise stated. The following examples illustrate the methodsand products of the present invention. Suitable modifications andadaptations of the described conditions and parameters normallyencountered in the art of molecular biology that are apparent to thoseskilled in the art are within the spirit and scope of the presentinvention.

In general, the practice of the present invention employs, unlessotherwise indicated, conventional techniques of chemistry, molecularbiology, recombinant DNA technology, immunology (especially, forexample, antibody technology), and standard techniques inelectrophoresis. See, for example, Sambrook, Fritsch and Maniatis,Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); AntibodyEngineering Protocols (Methods in Molecular Biology), 510, Paul, S.,Humana Pr (1996); Antibody Engineering: A Practical Approach (PracticalApproach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: ALaboratory Manual, Harlow et al., C.S.H.L. Press, Pub. (1999); andCurrent Protocols in Molecular Biology, eds. Ausubel et al., John Wiley& Sons (1992).

Example 1 Antibody Design

The methods described herein were used to obtain an optimized antibody(or an antigen-binding fragment thereof), although other methods knownto those skilled in the art can also be used without limitation. Basedon a computational analysis, positions can be identified within anygiven antibody where there is a difference (the larger the difference,the more significant it can be) between the charge distribution in anoptimized antibody-antigen complex and that in an originalantibody-antigen complex. Such differences in charge distribution arealso associated with changes in binding free energy of the antibody whenbound to the antigen in a solvent. The amino acid residue at such aposition can then be changed so that the electrostatic forces in theoriginal antibody more nearly approach (or in alternative embodiments,are more divergent from) those in the optimized antibody, therebymodulating binding free energy of the antibody when bound to an antigenin a solvent. Changes to the antibody are introduced according to a setof discrete criteria or rules as described herein and as described inInternational Publication Serial No. PCT/US04/24200, which isincorporated herein by reference in its entirety.

1. Rules for Modifying Antibodies for Improved Function

The rules for modifying antibodies were applied as follows. To modulatethe antigen-binding affinity of an antibody, for example, to improve orrestore such binding, basic sequence and/or structural data is firstacquired. Electrostatic charge optimization techniques were then appliedto suggest improved-affinity mutants. Typically, an electrostatic chargeoptimization was first used to determine the position(s) of the CDRresidue(s) that are sub-optimal for binding (Lee et al., J. Chem. Phys.,1997, 106, 8681-8690; Kangas et al., J. Chem. Phys., 1998, 109,7522-7545). Then, one or more CDR mutations (i.e., modifications) aresubjected to further computational analysis. Based on thesecalculations, the binding affinity was then determined for a subset ofmodified antibodies having one or more modifications according to therules of the invention.

Using a continuum electrostatics model, an electrostatic chargeoptimization was performed on each side chain of the amino acids in theCDRs of the antibody. A charge optimization gives charges at atomcenters but does not always yield actual mutation(s). Accordingly, around of charge optimizations was performed with various constraintsimposed to represent natural side chain characteristics at the positionsof interest. For example, an optimization was performed for a net sidechain charge of −1, 0, and +1 with the additional constraint that noatom's charge exceeded a particular value, for example, 0.85 electroncharge units. Candidate amino acid side chain positions, and residuemodifications at these positions, were then determined based on thepotential gain in electrostatic binding free energy observed in theoptimizations.

Binding free energy difference (in kcal/mol) in going from the nativeresidue to a completely uncharged sidechain isostere, such as, a residuewith the same shape but no charges or partial charges on the atoms werecalculated. Negative numbers indicate a predicted increase of bindingaffinity. Optimal charge distribution wherein the net side chain chargeis +1, 0, or −1 was used to calculate the binding free energydifference.

In those instances in which binding free energy difference is favorable(ΔG<−0.25 kcal/mol) and associated with a transition from the nativeresidue to a completely uncharged side chain isostere, such as, aresidue with the same shape but no charges or partial charges on theatoms, modifications from the set of amino acids with nonpolarsidechains, for example, Ala, Cys, Ile, Leu, Met, Phe, Pro, Val wereselected.

Where the binding free energy difference that can be obtained with anoptimal charge distribution in the side chain and a net side chaincharge of −1 is favorable (ΔG<−0.25 kcal/mol), modifications from theset of amino acids with negatively charged side chains, for example,Asp, Glu were selected.

Similarly, where the binding free energy difference that can be obtainedwith an optimal charge distribution in the side chain and a net sidechain charge of +1 is favorable (ΔG<−0.25 kcal/mol), modifications fromthe set of amino acids with positively charged sidechains, for example,Arg, His, Lys were selected.

Finally, in those cases where the binding free energy difference thatcan be obtained with an optimal charge distribution in the side chainand a net side chain charge of 0 is favorable (ΔG<−0.25 kcal/mol),modifications from the set of amino acids with uncharged polarsidechains, for example, Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr,Trp, Tyr, to which are added Cys, Gly, Met and Phe were selected.

As described herein, the designed modified antibodies were built insilico and the binding energy recalculated. Modified side chains werebuilt by performing a rotamer dihedral scan in CHARMM, using dihedralangle increments of 60 degrees, to determine the most desirable positionfor each side chain. Binding energies were then calculated for the wildtype (parent) and mutant (modified) complexes using thePoisson-Boltzmann electrostatic energy and additional terms for the vander Waals energy and buried surface area.

Results from these computational modification calculations were thenreevaluated as needed, for example, after subsequent reiterations of themethod either in silico or informed by additional experimentalstructural/functional data.

The rules allow for several predictions to be made which can becategorized as follows:

1) modifications at the interaction interface involving residues on theantibody that become partially buried upon binding (interactions areimproved by making hydrogen bonds with the antigen);

2) modifications of polar residues on the antibody that become buriedupon binding and thus pay a desolvation penalty but do not make anydirect electrostatic interactions with the antigen (improvements areusually made by modifying to a hydrophobic residue with similar shape tothe wild-type residue or by adding a residue that can make favorableelectrostatic interactions); and

3) modifications of surface residues on the antibody that are in regionsof uncomplimentary potentials. These modifications are believed toimprove long-range electrostatic interactions between the antibody andantigen without perturbing packing interactions at the bindinginterface.

The first type of modification is determined by inspection of basicphysical/chemical considerations, as these residues essentially makehydrogen bonds with unsatisfied hydrogen partners of the antigen. Unlikeother methods, the rules of the invention allowed for surprising residuemodifications in which the cost of desolvation is allowed to outweighthe beneficial interaction energy.

The second type of modification represents still another set ofmodifications, as the energy gained is primarily a result of eliminatingan unfavorable desolvation while maintaining non-polar interactions.

The third type of modification concerns long-range interactions thatshow potential for significant gain in affinity. These types ofmodifications are particularly interesting because they do not makedirect contacts with the antigen and, therefore, pose less of aperturbation in the delicate interactions at the antibody-antigeninterface.

Accordingly, when the desired side chain chemistries were determined forthe candidate amino acid position(s) according to the rules, the residueposition(s) was then modified or altered, for example, by substitution,insertion, or deletion, as further described herein.

In addition to the above rules for antibody modification, it is notedthat certain determinations, for example, solvent effects can befactored into initial (and subsequent) calculations of optimal chargedistributions.

2. Obtaining an Antibody or Antigen-Binding Fragment Thereof

The methods of the invention that are aimed at generating anon-naturally occurring antibody (or an antigen-binding fragmentthereof) can, but do not necessarily, begin by obtaining an antibody.That antibody may be referred to herein as a “parent” antibody orsometimes as a “first” antibody, and it can be used to obtaininformation that will allow one to modify or alter one or more aminoacid residues either within that antibody (such as, within the parentantibody) or within a modified or altered antibody having a sequencethat is similar to, or that contains portions of, the sequence of theparent antibody. As described herein, for example, one or more of theCDRs (or portions thereof) of a parent antibody, were replaced with thecorresponding CDR(s) of the modified antibody by standard geneticengineering techniques to accomplish the so-called CDR graft ortransplant. Accordingly, the method began with a mammalian monoclonal orpolyclonal antibody (for example, murine or primate), chimeric,CDR-grafted, humanized, or human antibody.

The parent antibodies were obtained from art-recognized sources orproduced according to art-recognized technologies. For example, theparent antibody was a CDR-grafted or humanized antibody having CDRregions derived from another source or species, for example, murine.

The parent antibody or any of the modified antibodies of the inventioncan also be in the format of a monoclonal antibody. Methods forproducing monoclonal antibodies are known in the art (see, for example,Kohler and Milstein, Nature, 1975, 256, 495-497), as well as techniquesfor stably introducing immunoglobulin-encoding DNA into myeloma cells(see, for example, Oi et al., Proc. Natl. Acad. Sci. USA, 1983, 80,825-829; Neuberger, EMBO J., 1983, 2, 1373-1378; and Ochi et al., Proc.Natl. Acad. Sci. USA, 1983, 80, 6351-6355). These techniques, whichinclude in vitro mutagenesis and DNA transfection, allow for theconstruction of recombinant immunoglobulins; these techniques can beused to produce the parent and modified antibodies used in the methodsof the invention or to produce the modified antibodies that result fromthose methods. Alternatively, the parent antibodies can be obtained froma commercial supplier. Antibody fragments (scFvs and Fabs) can also beproduced in E. coli (production methods and cellular hosts are describedfurther below).

The parent antibody or any of the modified antibodies of the inventioncan be an antibody of the IgA, IgD, IgE, IgG, or IgM class.

As noted above, the methods of the invention can be applied to more thanjust tetrameric antibodies (for example, antibodies having the structureof an immunoglobulin of the G class (an IgG)). For example, the methodsof modifying an antibody can be carried out with antigen-bindingfragments of any antibody as well. The fragments can be recombinantlyproduced and engineered, synthesized, or produced by digesting anantibody with a proteolytic enzyme. For example, the fragment can be anFab fragment; digestion with papain breaks the antibody at the region,before the inter-chain (such as, V_(H)-V_(H)) disulphide bond, thatjoins the two heavy chains. This results in the formation of twoidentical fragments that contain the light chain and the V_(H) andC_(H)1 domains of the heavy chain. Alternately, the fragment can be anF(ab′)₂ fragment. These fragments can be created by digesting anantibody with pepsin, which cleaves the heavy chain after theinter-chain disulphide bond, and results in a fragment that containsboth antigen-binding sites. Yet another alternative is to use a “singlechain” antibody. Single-chain Fv (scFv) fragments can be constructed ina variety of ways. For example, the C-terminus of V_(H) can be linked tothe N-terminus of V_(L). Typically, a linker (for example, (GGGGS)₄ (SEQID NO: 58)) is placed between V_(H) and V_(L). However, the order inwhich the chains can be linked can be reversed, and tags that facilitatedetection or purification (for example, Myc-, His-, or FLAG-tags) can beincluded (tags such as these can be appended to any antibody or antibodyfragment of the invention; their use is not restricted to scFv).Accordingly, and as noted below, tagged antibodies are within the scopeof the present invention. In alternative embodiments, the antibodiesused in the methods described herein, or generated by those methods, canbe heavy chain dimers or light chain dimers. Still further, an antibodylight or heavy chain, or portions thereof, for example, a single domainantibody (DAb), can be used.

As the methods of the invention can be iterative, the parent antibodymay not be a naturally occurring antibody. As the process of modifyingan antibody can be repeated as many times as necessary, the startingantibody (or antigen-binding fragment thereof) can be wholly non-humanor an antibody containing human FRs and non-human (for example, murine)CDRs. That is, the “parent” antibody can be a CDR-grafted antibody thatis subjected to the methods of the invention in order to improve theaffinity of the antibody, such as, affinity mature the antibody. Asnoted above, the affinity may only be improved to the extent that it isabout the same as (or not significantly worse than) the affinity of thenaturally occurring human antibody (the FR-donor) for its antigen. Thus,the “parent” antibody may, instead, be an antibody created by one ormore earlier rounds of modification, including an antibody that containssequences of more than one species (for example, human FRs and non-humanCDRs). The methods of the invention encompass the use of a “parent”antibody that includes one or more CDRs from a non-human (for example,murine) antibody and the FRs of a human antibody. Alternatively, theparent antibody can be completely human.

Where the structure is available, of course, one may begin thecomputational analysis with that structure (rather than creating itagain).

3. The Method of the Invention Informed by Antibody-Antigen StructuralData

Proteins are known to fold into three-dimensional structures that aredictated by the sequences of their amino acids and by the solvent inwhich a given protein (or protein-containing complex) is provided. Thethree-dimensional structure of a protein influences its biologicalactivity and stability, and that structure can be determined orpredicted in a number of ways. Generally, empirical methods use physicalbiochemical analysis. Alternately, tertiary structure can be predictedusing model building of three-dimensional structures of one or morehomologous proteins (or protein complexes) that have a knownthree-dimensional structure. X-ray crystallography is perhaps thebest-known way of determining protein structure (accordingly, the term“crystal structure” may be used in place of the term “structure”), butestimates can also be made using circular dichroism, light scattering,or by measuring the absorption and emission of radiant energy. Otheruseful techniques include neutron diffraction and nuclear magneticresonance (NMR). All of these methods are known to those of ordinaryskill in the art, and they have been well described in standardtextbooks (see, for example, Physical Chemistry, 4th Ed., W. J. Moore,Prentiss-Hall, N.J., 1972, or Physical Biochemistry, K. E. Van Holde,Prentiss-Hall, N.J., 1971)) and numerous publications. Any of thesetechniques can be carried out to determine the structure of an antibody,or antibody-antigen-containing complex, which can then be analyzedaccording to the methods of the present invention and, for example, usedto inform one or more steps of the method of the invention.

Similarly, these and like methods can be used to obtain the structure ofan antigen bound to an antibody fragment, including a fragmentconsisting of, for example, a single-chain antibody, Fab fragment, andthe like. Methods for forming crystals of an antibody, an antibodyfragment, or scFv-antigen complex have been reported by, for example,van den Elsen et al. (Proc. Natl. Acad. Sci. USA, 1999, 96, 13679-13684,which is expressly incorporated by reference herein).

4. Computational Analysis

The basic computational formulae used in carrying out the methods of theinvention are provided in, for example, U.S. Pat. No. 6,230,102, thecontents of which are hereby incorporated by reference in the presentapplication in their entirety.

As noted above, antibodies are altered (or “modified”) according to theresults of a computational analysis of electrostatic forces between theantibody and an antigen to which it binds, preferably, in accordance tothe discrete criteria or rules of the invention described herein. Thecomputational analysis allows one to predict the optimal chargedistribution within the antibody, and one way to represent the chargedistribution in a computer system is as a set of multipoles.Alternately, the charge distribution can be represented by a set ofpoint charges located at the positions of the atoms of the antibody.Once a charge distribution is determined (preferably, an optimal chargedistribution), one can modify the antibody to match, or better match,that charge distribution.

The computational analysis can be mediated by a computer-implementedprocess that carries out the calculations described in U.S. Pat. No.6,230,102. The computer program is adapted herein to consider the realworld context of antigen-antibody binding (and unlike other methods,this methods of the invention take into account, for example, solvent,long-range electrostatics, and dielectric effects in the binding betweenan antibody and its antigen in a solvent). The process is used toidentify modifications to the antibody structure that will achieve acharge distribution on the “matured” antibody that minimizes theelectrostatic contribution to binding free energy between the maturedantibody and its antigen (compared to that of the unmodified (“starting”or “parent”) antibody. As is typical, the computer system (or device(s))that performs the operations described here (and in more detail in U.S.Pat. No. 6,230,102) will include an output device that displaysinformation to a user (for example, a CRT display, an LCD, a printer, acommunication device such as a modem, audio output, and the like). Inaddition, instructions for carrying out the method, in part or in whole,can be conferred to a medium suitable for use in an electronic devicefor carrying out the instructions. Thus, the methods of the inventionare amendable to a high throughput approach comprising software (forexample, computer-readable instructions) and hardware (for example,computers, robotics, and chips). The computer-implemented process is notlimited to a particular computer platform, particular processor, orparticular high-level programming language.

A useful process is set forth in, for example, U.S. Pat. No. 6,230,102,and a more detailed exposition is provided in, for example, Lee andTidor (J. Chem. Phys., 1997, 106, 8681-8690), each of which is expresslyincorporated herein by reference.

5. Structure-Based Computational Design Methods

An exemplary approach to improving the affinity of an antibody to itsantigen is by using structure-based computational design methods. Onesuch method is known as sidechain repacking using the dead-endelimination algorithm (see for example, Lasters et al., Protein Eng., 8,815-22; Looger et al., J. Mol. Biol., 307, 429-45; Dahiyat et al.,Protein Sci., 5, 895-903). In such a calculation, antibody residues aresimultaneously mutated computationally to any of the 20 naturallyoccurring amino acids and the resulting mutants are evaluated foraffinity computationally. The list of computationally generated mutantscan be sorted by calculated stability of the mutant in order to generatea list of variants that will be expressed experimentally. In thecalculations the protein backbone is allowed very little or noflexibility, which ensures that the designed mutants are predicted to bestable with the given CDR conformations. Thus, the computationalanalysis allows one to predict antibody mutations that will enhance theaffinity to its antigen.

6. Generation of Antibodies and Antigen-Binding Fragments Thereof

The selection, cloning, and manufacture of antibodies, for example,chimeric, humanized, monoclonal, and single-chain antibodies is welldescribed in the art. In addition, the humanization of hu5c8 mAb hasbeen described previously (see, Lederman S et al., J Exp Med. 1992 Apr.1; 175(4):1091-101 and Karpusas M et al, Structure (Camb). 2001 Apr. 4;9(4):321-9, respectively. This antibody is available from the ATCC(PTA-4931). The 5c8 antibody was stably expressed in NS0 myeloma cellsand purified by Protein A and gel filtration chromatography. SDS-PAGEand analytical gel filtration chromatography demonstrated that theprotein formed the expected disulfide linked tetramer. The single-chainantibodies of the invention were typically expressed in E. coli andimmunopurified using standard techniques.

7. 5c8 Fab Production

5c8 Fab was expressed by the bicistronic plasmid pBEF064. The firstcistron contains 354 nucleotides of the 5c8 heavy chain encoding the 118amino acid heavy chain variable region followed in frame by 306nucleotides encoding the first 102 amino acids of the human IgG1constant domain and 18 nucleotides encoding a 6 histidine tag (SEQ IDNO: 59). A second ribosome entry site is located 7 nucleotides after theend of the heavy chain cistron. The second cistron contains 333nucleotides encoding the 111 amino acid 5c8 light chain variable regionfollowed in frame by 321 nucleotides encoding the 107 amino acid lightchain constant domain. Expression was carried out in E. coli and wasdriven by the ara-BAD promoter and the heavy and light chains aredirected to the periplasmic space by the OmpA (heavy chain) and PhoA(light chain) periplasmic localization signals. The periplasmiclocalization signals were cleaved from the protein during periplasmicexport.

8. Binding Assays

Binding assays were typically performed using the KinExA™ kit. The assaywas carried out by passing a dilute solution of the antibody (orantigen-binding fragment) through the column provided in the kit, andsome of the antibody (or the antigen-binding fragment thereof) interactswith the antigen on the bead. The antibody (or the fragment) was thendetected with a secondary anti-human IgG heavy and light chain antibodyconjugated with the fluorescent dye Cy5 (Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa.). The concentration of the antibody(or the fragment) was set so that the signal from the fluorescent dye isproportional to the concentration of protein. To obtain the solutionphase affinity of the interaction, the antibody (or the fragment) wasmixed with a dilution series of soluble antigen. These proteins(antibody and antigen) were allowed to reach equilibrium during athree-hour incubation at room temperature or an overnight incubation at4° C. The mixture was flowed over the antigen-containing column, and thesignal was proportional to the amount of unbound antibody (or antibodyfragment) that remains in solution. The resulting data was plotted on alinear-log scale graph and fit to a quadratic curve by non-linearregression, which gives a value for the K_(D).

9. Binding Assay 5c8-CD154

An ELISA-based competitive binding assay was carried out. Anti c-myc mAbwas coated onto NUNC Maxisorb plates at 10 μg/mL in PBS for 2 hrs at RT.Serial dilutions of unlabeled 5c8 Fab (mutants or wildtype) were madeand mixed with equal volumes of fixed concentration (30 ng/ml) ofbiotin-labeled 5c8 Fab competitor, and added to the plate. After 2 hoursincubation at room temperature, the plate was washed and boundbiotin-labeled 5c8 Fab competitor was detected with streptavidin-HRP.Binding affinities were obtained from four parameter curve fits.

Example 2 Antibodies

1. Generation of Antibodies

The selection, cloning, and humanization of hu5c8 mAb have beendescribed previously. See Lederman S et al., J Exp Med. 1992 Apr. 1;175(4):1091-101 and Karpusas M et al, Structure (Camb). 2001 Apr. 4;9(4):321-9, respectively. The hu5c8 mAb hyridoma is available from theATCC(HB10916).

2. CD154 Binding Assay

A FACS-based competitive binding assay was carried out on huCD154+ D1.1cells available from the ATCC(CRL-10915). The binding of 0.1 mg/ml ofbiotinylated hu5c8 mAb to cell surface CD154 was competed withtitrations of hu5c8 mAb or other antibody. Cell-bound biotinylated hu5c8mAb was detected with streptavidin-phycoerytherin (PE) (BD-PharMingenSan Diego, Calif., USA). Relative binding affinities were inferred fromthe IC50 values of four parameter curve fits.

3. CD154-FcγR Bridging Assays

FcγR binding affinities were measured using assays based on the abilityof the antibody to form a “bridge” between antigen and a FcγR bearingcell (see below). The FcγRI (CD64) bridging assay was performed bycoating 96-well Maxisorb ELISA plates (Nalge-Nunc Rochester, N.Y., USA)overnight at 4° C. with 1 mg/ml recombinant soluble human CD154 (Biogen,Karpusas M et al. Structure. 1995 Dec. 15; 3(12):1426) in PBS and thenblocking with 1% BSA in PBS. Titrations of antibody were then be boundto CD154 for 30 minutes at 37° C., the plates were washed, and thebinding of fluorescently labeled U937 (CD64+) cells was measured. TheU937 cells were grown in RPMI medium with 10% FBS, 10 mM HEPES,L-glutamine, and penicillin/streptomycin, split 1:2, and activated forone day prior to the assay with 1000 units/ml of IFNγ to increase FcγRIexpression.

The FcγRIII (CD16) bridging assays were performed using a monolayer ofCD154-expressing Chinese Hamster Ovary (CHO) cells (Biogen) grown in96-well tissue culture plates (Corning Life Sciences Acton, Mass., USA),with measurement of the mAb-dependent binding of fluorescently labeledJurkat cells transfected with CD16 (gift of Dana Farber Institute,Boston, Mass., USA). The CHO-CD154+ cells were seeded into 96-wellplates at 1×10⁵ cells/ml and grown to confluency in α MEM with 10%dialyzed FBS, 100 nM methotrexate, L-glutamine, andpenicillin/streptomycin (all reagents from Gibco-BRL Rockville, Md.,USA). CD16+ Jurkat cells, growing in RPMI with 10% FBS, 400 mg/mlGeneticin, 10 mM HEPES, sodium pyruvate, L-glutamine, andpenicillin/streptomycin (all reagents from Gibco-BRL), were split 1:2one day prior to performing the assay.

In the assays for both the FcγRI and FcγRIII receptors, the Fcreceptor-bearing cells were labeled with2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethylester (BCECF-AM) (Molecular Probes Eugene, Oreg., USA) for 20 minutes at37° C. After washing to remove excess BCECF-AM, 1×10⁵ of the labeledcells were incubated in the assay for 30 minutes at 37° C. Unbound FcγR+cells were removed by washing several times and plates read on aCytofluor 2350 Fluorescent Microplate Reader (Millipore CorporationBedford, Mass., USA) with an excitation wavelength of 485 nm and anemission wavelength of 530 nm.

Example 3 Improving the Antigen-Binding Affinity of an Anti-CD154Antibody

In this example, methods for improving the binding affinity of anantibody against a therapeutically relevant antigen target, aredescribed.

An antibody against human CD154 (see, for example, Yamada et al.,Transplantation, 2002, 73, S36-9; Schonbeck et al., Cell. Mol. Life.Sci., 2001, 58, 4-43; Kirk et al., Philos. Trans. R. Soc. Lond. B. Sci.,2001, 356, 691-702; Fiumara et al., Br. J. Haematol., 2001, 113, 265-74;and Biancone et al., Int. J. Mol. Med., 1999, 3, 343-53) which is amember of TNF family of proteins involved in mediating immunologicalresponses, was raised by affinity maturation in mice. The 5c8 monoclonalantibody was developed from such studies and determined to inhibit thepathological processes mediated by CD154.

In an effort to increase the affinity 5c8/CD154 interaction,electrostatic charge optimization techniques were applied to a crystalstructure of the antibody-antigen complex in a two-level procedure tosuggest improved-affinity mutants. First, electrostatic chargeoptimization was used to determine the position(s) of the CDR residue(s)that are sub-optimal for binding (Lee and Tidor, J. Chem. Phys., 1997,106, 8681-8690; Kangas and Tidor, J. Chem. Phys., 1998, 109, 7522-7545).Second, a set of CDR mutations were determined for further computationalanalysis. Based on these calculations, the binding affinity wascomputationally determined for 23 modified antibodies having a singlemutation (i.e., 23 “single mutants”). It was predicted that 8 of thesingle mutants would be more favorable than wild-type antibody both interms of electrostatic energy, and in terms of full energy functionincluding a van der Waals energy term and a solvent accessible surfacearea term. These terms are unrelated to electrostatic forces, but theywere calculated to ensure that the designed mutations did not contactother residues and would not reduce the amount of buried surface areasignificantly; increased buried surface area in complex formation isusually beneficial (see the “Full Energy” column of the table below).

Based on results from a charge optimization, mutations were determinedfor computational analysis (the optimal charge distributions and designmutations that were closer to optimal than the current residue wereexamined; this process was done by inspection). A charge optimizationgave charges at atom centers but did not yield actual mutation. A roundof charge optimizations was performed with various constraints imposedto represent natural side chain characteristics. For example, anoptimization was performed for a net side chain charge of −1, 0, and +1with the additional constraint that no atom's charge exceeded anabsolute value of 0.85 electron charge units.

The crystal structure of the CD154/5c8 complex (PDB code: 119R) wasprepared using standard procedures for adding hydrogens with the programCHARMM (Accelrys, Inc., San Diego, Calif.). N-acetamide andN-methylamide patches were applied to the N termini and C-termini,respectively. Using a continuum electrostatics model, an electrostaticcharge optimization was performed on each side chain of the amino acidsin the CDRs of the ACQ2 antibody. Appropriate side chain mutations werethen determined based on the potential gain in electrostatic bindingenergy observed in the optimizations. Side chains were built byperforming a rotamer dihedral scan in CHARMM, using dihedral angleincrements of 60 degrees, to determine the most desirable position foreach side chain. Binding energies were then calculated for the wild typeand mutant complexes using the Poisson-Boltzmann electrostatic energyand additional terms for the van der Waals energy and buried surfacearea.

The crystal structure of the CD40 ligand complexed with the Fab fragmentof a humanized neutralizing antibody (5c8) was solved to 3.1 Å at a pHof 6.50. Since CD154 is naturally a trimer, there are three 5c8 Fabmolecules and 5 CD154 molecules in the complex. They form threeindependent CD154/5c8 interfaces in the complex. A zinc (ZN) atom wasbound to each of the 5c8 Fab and it was included into the calculation.Calculations were carried out independently for three interfaces and theamino acid substitutions that were found to be favorable over wild typefor all three sites were exploited.

The following table shows the optimization results obtained for CDRvariable loop 1 in the light chain of 5c8 for all three 5c8 molecules.The Mut (Mutation energy) column corresponds to the binding free energydifference (in kcal/mol) in going from the native residue to acompletely uncharged sidechain isostere, i.e., a residue with the sameshape but no charges or partial charges on the atoms. Negative numbersindicate a predicted increase of binding affinity. The Opt−1 columncorresponds to the binding free energy difference that can be obtainedwith an optimal charge distribution in the side chain and a net sidechain charge of −1. The columns Opt0 and Opt1 correspond to the bindingfree energy differences with optimal charges, the net charge being 0 and+1, respectively. Based on these results and the visual inspection ofthe structure, mutations are designed that could take advantage of thesebinding free energy improvements. For instance, the mutation from SER 31to VAL, which is an uncharged isostere, makes use of the predicted −1.23to −0.98 kcal/mol in the mutation energy. The mutation GLN 27 to GLUuses the −1.21 to −0.88 kcal/mol predicted maximal free energy gain fora mutation to a side chain with a net charge of −1.

TABLE 1 Optimization results obtained for 5c8 CDR light chain variableloop 1 Chain Residue Mut Opt-1 Opt0 Opt1 1L 24 ARG −0.11 0.17 −0.11−0.37 1L 26 SER −0.06 −0.59 −0.06 0.57 1L 27 GLN 0.21 −1.21 −0.95 −0.261L 28 ARG 0.11 −0.96 −0.71 −0.40 1L 30 SER −0.01 −0.14 −0.42 −0.47 1L 31SER −1.23 3.88 −2.16 −0.42 1L 32 SER 1.45 0.91 −0.65 −0.67 1L 33 THR−0.02 −0.66 −0.41 0.07 1L 34 TYR −0.25 −1.00 −1.10 −0.80 1L 35 SER −0.020.00 −0.11 0.04 1L 36 TYR 0.01 −0.95 −1.31 1.74 1L 38 HSD −0.15 −0.48−0.70 −0.62 2L 24 ARG −0.46 −1.04 −0.46 0.13 2L 26 SER −0.29 −1.60 −0.790.19 2L 27 GLN 0.26 −0.88 −0.41 0.35 2L 28 ARG −0.59 −0.94 −0.46 0.08 2L30 SER 0.08 −0.38 −0.55 −0.42 2L 31 SER −0.98 4.04 −1.89 −0.54 2L 32 SER0.74 2.31 −0.86 −0.87 2L 33 THR 0.00 −0.65 −0.38 0.09 2L 34 TYR −0.09−0.62 −0.48 −0.12 2L 35 SER 0.09 0.02 0.09 0.18 2L 36 TYR 0.10 −1.70−1.24 2.37 2L 38 HSD −0.23 −1.20 −1.17 −0.79 3L 24 ARG −0.35 −0.34 −0.35−0.35 3L 26 SER −0.27 −1.23 −0.53 0.27 3L 27 GLN 0.11 −1.07 −0.71 −0.083L 28 ARG −0.30 −0.85 −0.30 0.15 3L 30 SER 0.03 0.02 −0.29 −0.36 3L 31SER −1.06 4.02 −2.03 −0.90 3L 32 SER 0.82 1.18 −0.85 −1.05 3L 33 THR0.20 −0.32 −0.15 0.29 3L 34 TYR 0.09 −0.80 −0.74 −0.38 3L 35 SER 0.06−0.05 −0.10 −0.02 3L 36 TYR 0.04 −0.99 −1.30 1.66 3L 38 HSD −0.20 −0.46−0.76 −0.72As described above, the designed mutants were built in silico and thebinding energy was recalculated. Results from these computationalmutation calculations are shown below. Numbers represent change inbinding affinity from wild-type to the mutant (negative meaning mutantis more favorable). Energies for all three chains of 5c8 are given.

TABLE 2 Computational mutation calculations for 5c8 CDRs Chain MutantFull Energy Electrostatics 1H TYR33PHE 0.197 −2.741 1H ASN59ASP −0.995−2.548 1H ASN59LEU −1.294 −2.517 1L SER26ASP −0.703 −0.712 1L GLN27GLU−0.514 −0.357 1L SER31VAL 8.154 −1.739 1L THR33ASP −0.219 −0.916 1LTYR54GLU −0.999 −0.729 2H TYR33PHE 0.623 −2.726 2H ASN59ASP −0.218−2.885 2H ASN59LEU −1.116 −3.067 2L SER26ASP −1.333 −1.627 2L GLN27GLU−0.658 −0.395 2L SER31VAL 9.293 −0.832 2L THR33ASP −0.430 −1.359 2LTYR54GLU −1.012 −1.030 3H TYR33PHE 0.145 −1.979 3H ASN59ASP −0.837−2.267 3H ASN59LEU −1.179 −2.271 3L SER26ASP −0.540 −0.565 3L GLN27GLU−0.497 −0.342 3L SER31VAL 8.129 −1.284 3L THR33ASP −0.337 −0.676 3LTYR54GLU −1.123 −0.825As the results show, the computational process described above wassuccessfully implemented to predict affinity enhancing side chainmutations.

The first type of mutation was resolved by inspection, as these residuesessentially make hydrogen bonds with unsatisfied hydrogen partners ofthe antigen. Surprisingly, the cost of desolvation seemed to outweighthe beneficial interaction energy in most cases. The second type ofmutation represents a less intuitive type or set of mutations, as theenergy gained is primarily a result of eliminating an unfavorabledesolvation while maintaining non-polar interactions. The third mutationtype concerns long-range interactions that show potential forsignificant gain in affinity. These types of mutations are particularlyinteresting because they do not make direct contacts with the antigenand, therefore, pose less of a perturbation in the delicate interactionsat the antibody-antigen interface.

In accordance with the computational data obtained as described above,mutants of 5c8 (Fab fragments) were generated, and their affinitytowards CD154 was compared to the affinity of the wild type 5c8 Fab inELISA-based competitive binding assay and KinExA™ assay described above.Selected results of some of the mutants are shown in the table thatfollows. Where an affinity assay has been conducted, the results areshown as affinity fold changes over original 5c8 Fab (wild type).Numbers greater than 1.0 indicate increased affinity, numbers smallerthan 1.0 indicate decreased affinity.

TABLE 3 Observed affinity changes for 5c8 altered Fab antibody fragmentsELISA fold- HCVD¹ LCVD² improvement KinExa Sequence Heavy Chain SequenceLight Chain over K_(d) Mutant SEQ ID NO: Mutation SEQ ID NO: Mutationwildtype (pM) # 4 (wildtype) 1 (wildtype) 1.0 264 48 T30H 1 — 2.3 307 149 Y33W 1 — 7.0 169 2 4 — Y36W 420 3 50 S54N 1 — 2.0 298 4 4 — 27 T33W5.0 158 5 4 — 13 S26D 2.6 142 6 4 — 14 Q27E 6.3 81 7 4 — S31V 0.02 8 4 —T33D 0.22 9 4 — Y54E 0.06 10 Y33F 1 — 0.05 11 N59D 1 — 0.13 12 N59L 1 —0.01 13 4 — 20 S26D/Q27E 5.6 41 14 4 — 32 S26D/Q27E/T33W 3.8 44 15 48T30H 20 S26D/Q27E 5.8 70 16 49 Y33W 32 S26D/Q27E/T33W 5.9 23 17 48 T30H32 S26D/Q27E/T33W 5.4 54 18 50 S54N 20 S26D/Q27E 65 19 50 S54N 32S26D/Q27E/T33W 44 20 55 T30H/Y33W/S54N 20 S26D/Q27E 101 21 55T30H/Y33W/S54N 32 S26D/Q27E/T33W 55 22 49 Y33W 20 S26D/Q27E 5.2 53 23 54T30H/Y33W 20 S26D/Q27E 8.0 52 24 54 T30H/Y33W 32 S26D/Q27E/T33W 5.6 1225 56 T30H/S54N 32 S26D/Q27E/T33W 63 26 57 Y33W/S54N 32 S26D/Q27E/T33W52 27 57 Y33W/S54N 20 S26D/Q27E 77 28 ¹HCVD stands for Heavy ChainVariable Domain ²LCVD stands for Light Chain Variable Domain

Analysis of the deviations from the ideal charge distributions on the 50amino acids in the CDRs of the 5c8 antibody resulted in prediction offive mutants that would have higher affinity towards CD154 compared towild-type: Asn59Asp, Asn59Leu, Ser26Asp, Gln27G1u, and Tyr54Glu. Thesemutants were constructed and characterized by competitive ELISA. Theimproved affinity mutants S26D and Q27E were also combined into a doublemutant that had a 9-fold higher affinity compared to the wildtype 5c8Fab.

The side chain repacking algorithm was applied to the CDRs of 5c8. For30 CDR residues, 10⁴⁰ sequences were computationally analyzed and 14single and double mutants were selected for experimental follow-up.Several mutants showed higher affinity compared to wildtype 5c8 Fab. Theside chain repacking mutants were further combined with higher affinityelectrostatic mutants. The combination 5 point mutation had 25-foldhigher affinity compared to the wild type. Furthermore, in cells itshowed superior inhibition of ICAM upregulation (biological consequenceof CD40-CD154 interaction) which was comparable by blocking exhibited bya full length 5c8 antibody. An overall hit rate of 40% in mutantprediction was achieved for both methods.

A T cell-dependent B cell activation assay was performed Inhibition of Tcell-dependent B cell activation was measured in D1.1: Ramosco-cultures. Engagement of CD40 on B cells by CD154 results in B cellactivation and upregulation of ICAM-1 (CD54). Anti-CD154 mAbs and Fabsinhibit the B cell activation by disrupting the CD40-CD154 interaction.Briefly, titrations of anti-CD154 Fab were added to a 1:4 ratio ofD1.1:Ramos cells and incubated overnight. Two-color FACS staining(CD20FITC/CD54APC), which allows for quantitation of ICAM-1 upregulationon the Ramos cells, was performed. Results are shown in Table 4.

TABLE 4 Observed potencies in T cell-dependent B cell activation assayHeavy Chain Light Chain Mutation Mutation IC50 (ug/ml) (wildtype)(wildtype) 1.215 — S26D/Q27E 0.287 Y33W S26D/Q27E/T33W 0.131 T30H/Y33WS26D/Q27E/T33W 0.165

EQUIVALENTS

For one skilled in the art, using no more than routine experimentation,there are many equivalents to the specific embodiments of the inventiondescribed herein. Such equivalents are intended to be encompassed by thefollowing claims.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference (including, but not limitedto, journal articles, U.S. and non-U.S. patents, patent applicationpublications, international patent application publications, gene bankaccession numbers, and the like) cited in the present application isincorporated herein by reference in its entirety.

1.-97. (canceled)
 98. An antibody or antigen binding fragment thereofthat binds CD154 and that comprises a light chain variable region, whichcomprises light chain CDRs VL CDR1, VL CDR2, and VL CDR3, and a heavychain variable region, which comprises heavy chain CDRs VH CDR1, VHCDR2, and VH CDR3, wherein (i) VL CDR1, VL CDR2, and VL CDR3respectively have the sequences of (a) amino acids 24-38, 54-60, and93-101 of SEQ ID NO:1 or (b) amino acids 24-38, 54-60, and 93-101 of SEQID NO:1 except that one or a combination of substitutions is present,wherein the substitutions are selected from the group consisting of Serat position 26 substituted with Asp, Gln at position 27 substituted withGlu, and Thr at position 33 substituted with Trp; and (ii) VH CDR1, VHCDR2, and VH CDR3 respectively have the sequences of (a) amino acids26-35, 50-66, and 99-107 of SEQ ID NO:4 or (b) amino acids 26-35, 50-66,and 99-107 of SEQ ID NO:4 except that one or a combination ofsubstitutions is present, wherein the substitutions are selected fromthe group consisting of Thr at position 30 substituted with His, Tyr atposition 33 substituted with Trp, and Ser at position 54 substitutedwith Asn; wherein when the light chain CDR sequences have the wild type5c8 CDR sequences according to (i)(a) above, the heavy chain CDRsequences are not the wild type 5c8 CDR sequences according to (ii)(a)above.
 99. The antibody or antigen binding fragment of claim 98, whereinVL CDR1, VL CDR2, and VL CDR3 respectively have the sequences of aminoacids 24-38, 54-60, and 93-101 of SEQ ID NO:1.
 100. The antibody orantigen binding fragment of claim 98, wherein VH CDR1, VH CDR2, and VHCDR3 respectively have the sequences of amino acids 26-35, 50-66, and99-107 of SEQ ID NO:4.
 101. The antibody or antigen binding fragment ofclaim 98, wherein VL CDR1, VL CDR2, and VL CDR3 respectively have thesequences of amino acids 24-38, 54-60, and 93-101 of a sequence selectedfrom SEQ ID NO:27, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:20, or SEQ IDNO:32.
 102. An antibody or antigen binding fragment of claim 98, whereinVH CDR1, VH CDR2, and VH CDR3 respectively have the sequences of aminoacids 26-35, 50-66, and 99-107 of a sequence selected from SEQ ID NO:48,SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:55, SEQ ID NO:54, SEQ ID NO:56, orSEQ ID NO:57.
 103. The antibody or antigen binding fragment of claim 98,wherein the antibody or antigen-binding fragment binds to CD 154 withgreater affinity than does an antibody or antigen binding fragment thatis otherwise identical but has wild type 5c8 CDR sequences VL CDR1, VLCDR2, and VL CDR3 that respectively have the sequences of amino acids24-38, 54-60, and 93-101 of SEQ ID NO:1 and VH CDR1, VH CDR2, and VHCDR3 that respectively have the sequences of amino acids 26-35, 50-66,and 99-107 of SEQ ID NO:4.
 104. The antibody or antigen binding fragmentof claim 103, wherein the affinity is assessed using ELISA or a KinExA™assay.
 105. The antibody or antigen binding fragment of claim 98,wherein the antibody or antigen-binding fragment shows improved potencyin inhibiting T cell-dependent B cell activation compared with anantibody, or antigen binding fragment, that that is otherwise identicalbut has wild type 5c8 CDR sequences VL CDR1, VL CDR2, and VL CDR3 thatrespectively have the sequences of amino acids 24-38, 54-60, and 93-101of SEQ ID NO:1 and VH CDR1, VH CDR2, and VH CDR3 that respectively havethe sequences of amino acids 26-35, 50-66, and 99-107 of SEQ ID NO:4.106. The antigen-binding fragment of claim 98, which is selected fromthe group consisting of a single chain antibody (scFv), a F(ab′)₂fragment, a Fab fragment, and an Fd fragment.
 107. The antibody orantigen-binding fragment of claim 98, labeled with a detectable marker.108. The antibody or antigen-binding fragment of claim 98, conjugated toa therapeutic agent or a bead.
 109. The antibody or antigen-bindingfragment of claim 98, comprising at least one high molecular-weightpolymer.
 110. The antibody or antigen-binding fragment of claim 98,wherein at least one amino acid of the antibody or antigen-bindingfragment is PEGylated or glycosylated.
 111. A composition comprising theantibody or antigen-binding fragment of claim
 98. 112. A kit comprisingthe antibody or antigen-binding fragment of claim
 98. 113. A method oftreating a human disease or disorder comprising administering atherapeutically effective amount of the antibody or antigen-bindingfragment of claim 98 to a human having the disease or disorder, suchthat the disease or disorder is diminished, wherein the disease ordisorder is inflammation, Myasthenia gravis, Graves' disease, idiopathicthrombocytopenia purpura, hemolytic anemia, diabetes mellitus, Crohn'sdisease, multiple sclerosis, a drug-induced autoimmune disease,rejection of a transplanted organ, graft-vs-host disease, an allergicresponse, an autoimmune response, fibrosis, gastrointestinal disease,vascular disease, a T cell cancer, or viral infection of T cells by theHTLV I virus.
 114. An antibody or antigen binding fragment thereof thatbinds CD154 and that comprises a light chain variable domain comprisinglight chain CDRs VL CDR1, VL CDR2, and VL CDR3, and a heavy chainvariable domain comprising heavy chain CDRs VH CDR1, VH CDR2, and VHCDR3, wherein (i) VL CDR1, VL CDR2, and VL CDR3 respectively have thesequences of amino acids 24-38, 54-60, and 93-101 of SEQ ID NO:1 and VHCDR1, VH CDR2, and VH CDR3 respectively have the sequences of aminoacids 26-35, 50-66, and 99-107 of SEQ ID NO:48; (ii) VL CDR1, VL CDR2,and VL CDR3 respectively have the sequences of amino acids 24-38, 54-60,and 93-101 of SEQ ID NO:1 and VH CDR1, VH CDR2, and VH CDR3 respectivelyhave the sequences of amino acids 26-35, 50-66, and 99-107 of SEQ IDNO:49; (iii) VL CDR1, VL CDR2, and VL CDR3 respectively have thesequences of amino acids 24-38, 54-60, and 93-101 of SEQ ID NO:1 and VHCDR1, VH CDR2, and VH CDR3 respectively have the sequences of aminoacids 26-35, 50-66, and 99-107 of SEQ ID NO:50; (iv) VL CDR1, VL CDR2,and VL CDR3 respectively have the sequences of amino acids 24-38, 54-60,and 93-101 of SEQ ID NO:27 and VH CDR1, VH CDR2, and VH CDR3respectively have the sequences of amino acids 26-35, 50-66, and 99-107of SEQ ID NO:4; (v) VL CDR1, VL CDR2, and VL CDR3 respectively have thesequences of amino acids 24-38, 54-60, and 93-101 of SEQ ID NO:13 and VHCDR1, VH CDR2, and VH CDR3 respectively have the sequences of aminoacids 26-35, 50-66, and 99-107 of SEQ ID NO:4; (vi) VL CDR1, VL CDR2,and VL CDR3 respectively have the sequences of amino acids 24-38, 54-60,and 93-101 of SEQ ID NO:14 and VH CDR1, VH CDR2, and VH CDR3respectively have the sequences of amino acids 26-35, 50-66, and 99-107of SEQ ID NO:4; (vii) VL CDR1, VL CDR2, and VL CDR3 respectively havethe sequences of amino acids 24-38, 54-60, and 93-101 of SEQ ID NO:20and VH CDR1, VH CDR2, and VH CDR3 respectively have the sequences ofamino acids 26-35, 50-66, and 99-107 of SEQ ID NO:4; (viii) VL CDR1, VLCDR2, and VL CDR3 respectively have the sequences of amino acids 24-38,54-60, and 93-101 of SEQ ID NO:32 and VH CDR1, VH CDR2, and VH CDR3respectively have the sequences of amino acids 26-35, 50-66, and 99-107of SEQ ID NO:4; (ix) VL CDR1, VL CDR2, and VL CDR3 respectively have thesequences of amino acids 24-38, 54-60, and 93-101 of SEQ ID NO:20 and VHCDR1, VH CDR2, and VH CDR3 respectively have the sequences of aminoacids 26-35, 50-66, and 99-107 of SEQ ID NO:48; (x) VL CDR1, VL CDR2,and VL CDR3 respectively have the sequences of amino acids 24-38, 54-60,and 93-101 of SEQ ID NO:32 and VH CDR1, VH CDR2, and VH CDR3respectively have the sequences of amino acids 26-35, 50-66, and 99-107of SEQ ID NO:49; (xi) VL CDR1, VL CDR2, and VL CDR3 respectively havethe sequences of amino acids 24-38, 54-60, and 93-101 of SEQ ID NO:32and VH CDR1, VH CDR2, and VH CDR3 respectively have the sequences ofamino acids 26-35, 50-66, and 99-107 of SEQ ID NO:48; (xii) VL CDR1, VLCDR2, and VL CDR3 respectively have the sequences of amino acids 24-38,54-60, and 93-101 of SEQ ID NO:20 and VH CDR1, VH CDR2, and VH CDR3respectively have the sequences of amino acids 26-35, 50-66, and 99-107of SEQ ID NO:50; (xiii) VL CDR1, VL CDR2, and VL CDR3 respectively havethe sequences of amino acids 24-38, 54-60, and 93-101 of SEQ ID NO:32and VH CDR1, VH CDR2, and VH CDR3 respectively have the sequences ofamino acids 26-35, 50-66, and 99-107 of SEQ ID NO:50; (xiv) VL CDR1, VLCDR2, and VL CDR3 respectively have the sequences of amino acids 24-38,54-60, and 93-101 of SEQ ID NO:20 and VH CDR1, VH CDR2, and VH CDR3respectively have the sequences of amino acids 26-35, 50-66, and 99-107of SEQ ID NO:55; (xv) VL CDR1, VL CDR2, and VL CDR3 respectively havethe sequences of amino acids 24-38, 54-60, and 93-101 of SEQ ID NO:32and VH CDR1, VH CDR2, and VH CDR3 respectively have the sequences ofamino acids 26-35, 50-66, and 99-107 of SEQ ID NO:55; (xvi) VL CDR1, VLCDR2, and VL CDR3 respectively have the sequences of amino acids 24-38,54-60, and 93-101 of SEQ ID NO:20 and VH CDR1, VH CDR2, and VH CDR3respectively have the sequences of amino acids 26-35, 50-66, and 99-107of SEQ ID NO:49; (xvii) VL CDR1, VL CDR2, and VL CDR3 respectively havethe sequences of amino acids 24-38, 54-60, and 93-101 of SEQ ID NO:20and VH CDR1, VH CDR2, and VH CDR3 respectively have the sequences ofamino acids 26-35, 50-66, and 99-107 of SEQ ID NO:54; (xviii) VL CDR1,VL CDR2, and VL CDR3 respectively have the sequences of amino acids24-38, 54-60, and 93-101 of SEQ ID NO:32 and VH CDR1, VH CDR2, and VHCDR3 respectively have the sequences of amino acids 26-35, 50-66, and99-107 of SEQ ID NO:54; (ixx) VL CDR1, VL CDR2, and VL CDR3 respectivelyhave the sequences of amino acids 24-38, 54-60, and 93-101 of SEQ IDNO:32 and VH CDR1, VH CDR2, and VH CDR3 respectively have the sequencesof amino acids 26-35, 50-66, and 99-107 of SEQ ID NO:56; (xx) VL CDR1,VL CDR2, and VL CDR3 respectively have the sequences of amino acids24-38, 54-60, and 93-101 of SEQ ID NO:32 and VH CDR1, VH CDR2, and VHCDR3 respectively have the sequences of amino acids 26-35, 50-66, and99-107 of SEQ ID NO:57; (xxi) VL CDR1, VL CDR2, and VL CDR3 respectivelyhave the sequences of amino acids 24-38, 54-60, and 93-101 of SEQ IDNO:20 and VH CDR1, VH CDR2, and VH CDR3 respectively have the sequencesof amino acids 26-35, 50-66, and 99-107 of SEQ ID NO:57.
 115. Theantibody or antigen binding fragment of claim 114, wherein the antibodyor antigen-binding fragment binds to CD 154 with greater affinity thandoes an antibody or antigen binding fragment that is otherwise identicalbut has wild type 5c8 CDR sequences VL CDR1, VL CDR2, and VL CDR3 thatrespectively have the sequences of amino acids 24-38, 54-60, and 93-101of SEQ ID NO:1 and VH CDR1, VH CDR2, and VH CDR3 that respectively havethe sequences of amino acids 26-35, 50-66, and 99-107 of SEQ ID NO:4.116. The antibody or antigen binding fragment of claim 115, wherein theaffinity is assessed using ELISA or a KinExA™ assay.
 117. The antibodyor antigen binding fragment of claim 114, wherein the antibody orantigen-binding fragment shows improved potency in inhibiting Tcell-dependent B cell activation compared with an antibody, or antigenbinding fragment, that that is otherwise identical but has wild type 5c8CDR sequences VL CDR1, VL CDR2, and VL CDR3 that respectively have thesequences of amino acids 24-38, 54-60, and 93-101 of SEQ ID NO:1 and VHCDR1, VH CDR2, and VH CDR3 that respectively have the sequences of aminoacids 26-35, 50-66, and 99-107 of SEQ ID NO:4.
 118. The antigen-bindingfragment of claim 115, which is selected from the group consisting of asingle chain antibody (scFv), a F(ab′)₂ fragment, a Fab fragment, and anFd fragment.
 119. The antibody or antigen-binding fragment of claim 115,labeled with a detectable marker.
 120. The antibody or antigen-bindingfragment of claim 115, conjugated to a therapeutic agent or a bead. 121.The antibody or antigen-binding fragment of claim 115, comprising atleast one high molecular-weight polymer.
 122. The antibody orantigen-binding fragment of claim 115, wherein at least one amino acidof the antibody or antigen-binding fragment is PEGylated orglycosylated.
 123. A composition comprising the antibody orantigen-binding fragment of claim
 115. 124. A kit comprising theantibody or antigen-binding fragment of claim
 115. 125. A method oftreating a human disease or disorder comprising administering atherapeutically effective amount of the antibody or antigen-bindingfragment of claim 115 to a human having the disease or disorder, suchthat the disease or disorder is diminished, wherein the disease ordisorder is inflammation, Myasthenia gravis, Graves' disease, idiopathicthrombocytopenia purpura, hemolytic anemia, diabetes mellitus, Crohn'sdisease, multiple sclerosis, a drug-induced autoimmune disease,rejection of a transplanted organ, graft-vs-host disease, an allergicresponse, an autoimmune response, fibrosis, gastrointestinal disease,vascular disease, a T cell cancer, or viral infection of T cells by theHTLV I virus.
 126. A nucleic acid comprising a nucleotide sequenceencoding the light or heavy chain CDRs of an antibody, or antigenbinding fragment, according to claim 98.